CN101631796B - Compound having structure derived from mononucleoside or mononucleotide, nucleic acid, labeling substance, and method and kit for detection of nucleic acid - Google Patents

Abstract

The present invention provides labels capable of efficiently detecting, for example, nucleic acid double helix structures. A compound having a structure derived from a mononucleoside or a mononucleotide, a tautomer or a stereoisomer thereof, or a salt thereof is provided, wherein the structure is represented by the following formula (1), (1b) or (1c) said. For example, B is an atomic group with a nucleic acid base skeleton, E is an atomic group with a deoxyribose skeleton, a ribose skeleton or a structure derived therefrom, or an atomic group with a peptide structure or a peptidomimetic structure, and Z ¹¹ and Z ¹² represent hydrogen atoms respectively , a protecting group, or an atomic group exhibiting fluorescence may be the same or different.

Description

Compounds, nucleic acids, markers, and nucleic acid detection methods and kits having structures derived from single nucleosides or single nucleotides

technical field

The present invention relates to a compound, a nucleic acid, a marker, a nucleic acid detection method and a kit having a structure derived from a single nucleoside or a single nucleotide.

Background technique

In genetic diagnosis of diseases and gene expression analysis, etc., it is necessary to detect nucleic acids having a specific sequence. Methods using fluorescence are often used for this purpose, for example fluorescent probes obtained by covalently binding a fluorescent dye to DNA are commonly used as labels.

A problem with such labels (fluorescent probes) is that they fluoresce, for example without forming a double helix with a complementary nucleic acid. The method of using FRET to eliminate the fluorescence of the probe itself is effective (Non-Patent Documents 1-4, etc.), but there are problems such as the cost of introducing two kinds of fluorescent dyes.

In addition, thiazole orange is known as one of cyanine dyes that are fluorescent dyes that increase fluorescence intensity through interaction with DNA or RNA. Although an example of making a fluorescent probe by covalently bonding thiazole orange to DNA has been attempted, it still emits strong fluorescence through the interaction with single-stranded DNA containing purine bases (Non-Patent Document 5), so in The increase in fluorescence intensity upon double helix formation was small and could not be said to be successful (Non-Patent Documents 6 and 7).

Non-Patent Document 1: Tyagi, S. Kramer, F.R. (1996) Nat. Biotechnol. 14, 303-308.

Non-Patent Document 2: Nazarenko, I.A., Bhatnagar, S.K., Hohman, R.J. (1997) Nucleic Acids Res. 25, 2516-2521.

Non-Patent Document 3: Gelmini, S., Orlando, C., Sestini, R., Vona, G., Pinzani, P., Ruocco, L., Pazzagli, M. (1997) Clin.Chem.43, 752- 758.

Non-Patent Document 4: Whitcombe, D., Theaker, J., Buy, S.P., Brown, T., Little, S. (1999) Nat. Biotechnol. 17, 804-807.

Non-Patent Document 5: Biopolymers 1998, 46, 39-51.

Non-Patent Document 6: Analytica Chimica Acta 2002, 470, 57-70.

Non-Patent Document 7: Chemistry-A European Journal 2006, 12, 2270-2281.

Contents of the invention

Therefore, an object of the present invention is to provide a label capable of efficiently detecting the double helix structure of nucleic acid.

In order to solve the above problems, the compound of the present invention is a compound having a structure derived from a mononucleoside or a mononucleotide, a tautomer or a stereoisomer thereof, or a salt thereof, wherein the structure is represented by the following formula ( 1), (1b) or (1c).

[chemical 20]

In said formulas (1), (1b) and (1c),

B is an atomic group with a natural nucleic acid base (adenine, guanine, cytosine, thymine or uracil) skeleton or an artificial nucleic acid base skeleton,

E is

(i) a radical having a deoxyribose backbone, a ribose backbone, or a structure derived from either, or

(ii) radicals having a peptide structure or a peptidomimetic structure,

Z ¹¹ and Z ¹² respectively represent a hydrogen atom, a protecting group or an atomic group exhibiting fluorescence, which may be the same or different,

Q in

When E is the atomic group of (i) above, it is O,

When E is the atomic group of (ii) above, it is NH,

X in

When E is the atomic group of the aforementioned (i), it is a hydrogen atom, a hydroxyl protecting group that can be deprotected with an acid, a phosphoric acid group (monophosphate group), a diphosphate group (diphosphate group) or a triphosphate group (triphosphate group). base),

When E is the atomic group of the aforementioned (ii), it is a hydrogen atom or an amino protecting group,

Y in

When E is the atomic group of the aforementioned (i), it is a hydrogen atom, a hydroxyl protecting group or a phosphoramidite group,

When E is the atomic group of (ii) above, it is a hydrogen atom or a protecting group,

L ¹ , L ² and L ³ are each a linker (bridging atom or atomic group), the length of the main chain (the number of atoms in the main chain) is arbitrary, and in the main chain, each may contain or not contain C, N, O, S , P and Si, in the main chain, each may contain or not contain single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, imine group, ether bond, thioether bond and thioester bond , L ¹ , L ² and L ³ can be the same or different from each other,

D is CR, N, P, P=O, B or SiR, R is a hydrogen atom, an alkyl group or any substituent,

b is a single bond, double bond or triple bond,

Alternatively, in the formula (1), L ¹ and L ² are the linkers, L ³ , D and b do not exist, and L ¹ and L ² are directly connected to B,

In said formula (1b), T is in

When E is the atomic group in (i) above, it is a phosphate bridge (PO ₄ ^- ), wherein one or more oxygen atoms (O) may be replaced by sulfur atoms (S),

When E is the atomic group of (ii) above, it is NH.

In addition, the nucleic acid of the present invention is a nucleic acid having at least one structure represented by the following formula (16), (16b), (17), (17b), (18) or (18b), its tautomer or stereo isomers, or salts thereof. In addition, in this specification, when the bonds in the chemical formulas (such as the following formulas (16), (16b), (17), (17b), (18) and (18b) extend from the inside of the brackets to the outside of the brackets and are outside the brackets When an asterisk is added to the bond, the asterisk indicates that a certain atom or atomic group is bonded to the bond.

[chem 21]

[chem 22]

[chem 23]

[chem 24]

[chem 25]

[chem 26]

In formulas (16), (16b), (17), (17b), (18) and (18b)

B, E, Z ¹¹ , Z ¹² , L ¹ , L ² , L ³ , D and b each have the same structure as in the above formula (1), (1b) or (1c),

in

In formulas (16), (17) and (18), E is the atomic group of (i) in the above formula (1), (1b) or (1c), at least one O atom in the phosphate bridge can be replaced by an S atom ,

In formulas (16b), (17b) and (18b), E is the atomic group of (ii) in the above-mentioned formulas (1), (1b) or (1c),

In formulas (17) and (17b), each B may be the same or different, and each E may be the same or different.

In addition, the markers of the present invention are

(i) Markers: two planar chemical structures in a molecule are not in the same plane, but exist at an angle, but when the molecule is inserted or grooved into a nucleic acid, the two planar chemical structures Arranged in the same plane to produce fluorescent light;

(ii) A label formed of two or more groups of dye molecules in which, although the excitonic effect due to parallel aggregation of two or more dye molecules does not exhibit fluorescence emission, these molecules are bound to nucleic acid at insertion or groove During the middle, the release of the aggregation state will produce fluorescence emission; or

(iii) A complex marker having a chemical structure having two or more dye molecules in the same molecule as a characteristic chemical structure, wherein although the excitonic effect due to the parallel aggregation of two or more dye molecules is not Fluorescence is exhibited, but upon intercalation or groove binding of these molecules into a nucleic acid, fluorescence occurs upon release of the aggregated state.

In addition, the nucleic acid detection method of the present invention is:

(1) nucleic acid detection method comprising the following steps:

Using the label of the present invention—labeled mononucleotide or labeled oligonucleotide as a substrate to carry out nucleic acid synthesis, thereby synthesizing a double-stranded nucleic acid inserted or groove-bonded with the fluorescent atomic group or dye molecular structure;

separately measuring the fluorescence intensity before and after the double-stranded nucleic acid synthesis step; and

Detecting nucleic acid synthesis by comparing the fluorescence intensity before and after the double-stranded nucleic acid synthesis step;

(II) A nucleic acid detection method comprising the following steps:

Using the marker of the present invention—single-stranded nucleic acid as the first nucleic acid, hybridizing it with a second nucleic acid having a sequence complementary to the first nucleic acid or a sequence similar to the complementary sequence to perform nucleic acid synthesis, thereby synthesizing the insertion A double-stranded nucleic acid with or groove-bonded with the atomic group exhibiting fluorescence or the molecular structure of the dye;

separately measuring the fluorescence intensity before and after the double-stranded nucleic acid synthesis step; and

Detecting hybridization between the first nucleic acid and the second nucleic acid by comparing the fluorescence intensity before and after the double-stranded nucleic acid synthesis step;

(III) A nucleic acid detection method comprising the following steps:

Using the marker of the present invention—single-stranded nucleic acid as the first nucleic acid, hybridizing it with a second nucleic acid having a sequence complementary to the first nucleic acid or a sequence similar to the complementary sequence to perform nucleic acid synthesis, thereby synthesizing the insertion A double-stranded nucleic acid with or groove-bonded with the atomic group exhibiting fluorescence or the molecular structure of the dye;

separately measuring the fluorescence intensity before and after the double-stranded nucleic acid synthesis step; and

Detecting hybridization between the first nucleic acid and the second nucleic acid by comparing the fluorescence intensity before and after the double-stranded nucleic acid synthesis step;

or

(IV) A nucleic acid detection method, characterized in that a third nucleic acid is used to detect the formation of a triple-stranded nucleic acid or a nucleic acid analog, and the third nucleic acid is obtained by using a nucleic acid having the first nucleic acid sequence, the second nucleic acid sequence, or Sequences complementary to these sequences, or sequences similar to sequences complementary to said sequences, are labeled or not labeled with the marker or complex marker of the present invention.

In addition, the kit of the present invention contains a nucleic acid synthesis device, a marker and a fluorescence intensity measurement device, and the marker is the marker of the present invention.

The compounds and nucleic acids of the present invention can be used as labels capable of efficiently detecting the double helix structure of nucleic acids because they have such structures. More specifically, in, for example, said formula (1), (1b), (1c), (16), (16b), (17), (17b), (18) or (18b), Z ¹¹ and Compounds or nucleic acids in which Z ¹² is an atomic group exhibiting fluorescence are suitable for use as the label of the present invention. In addition, compounds or nucleic acids in which Z ¹¹ and Z ¹² are hydrogen atoms or protecting groups can be used as synthetic raw materials or synthetic intermediates of the label. However, the use of the compound and nucleic acid of the present invention is not limited thereto, and may be used for any purpose.

Description of drawings

Figure 1: Figure 1 is a diagram schematically showing the principles of the present invention.

Figure 2: Figure 2 shows the ¹ H and ¹³ C NMR spectra of the example compounds.

Figure 3: Figure 3 shows the 1H and 13C NMR spectra of other compounds of the examples.

Figure 4: Figure 4 shows the MALDI TOF mass spectrum of purified DNA oligomer 5'-d(CGCAATXTAACGC)-3'. The arrow is the mass peak (4101.9) of the purified product. [MH] ^-calculated 4101.8 by molecular weight calculated 4102.8 (C ₁₃₄ H ₁₇₆ N ₅₂ O ₇₆ P ₁₂ ), which is consistent.

Figure 5: Figure 5 shows the MALDI TOF mass spectrum of the reaction product of DNA oligomer 5'-d(CGCAATXTAACGC)-3' and biotin derivative. The arrow is the mass peak (4554.3) of the purified product. [MH] ^-calculated 4554.4 by molecular weight calcd. 4555.4 (C ₁₃₄ H ₁₇₆ N ₅₂ O ₇₆ P ₁₂ ), which is consistent.

Figure 6: Figure 6 shows the ¹ H NMR spectrum (DMSO-d6) of the example compound (DNA labeled with a dye).

Figure 7: Figure 7 shows the reverse phase HPLC profile of the compound of Figure 6 (DNA labeled with a dye).

Figure 8: Figure 8 shows the MALDI TOF mass spectrum of the compound of Figure 6 (DNA labeled with a dye).

Fig. 9: Fig. 9 shows the UV spectra of three samples when the fluorescent probe of the embodiment is in a single-stranded state, a DNA-DNA double helix, and a DNA-RNA double helix.

Figure 10: Figure 10 shows the fluorescence spectra of three samples when the fluorescent probe in Figure 9 is in a single-stranded state, a DNA-DNA double helix, and a DNA-RNA double helix when an excitation light of 488 nm is used.

Figure 11: Figure 11 shows the fluorescence spectra of three samples when the fluorescent probe in Figure 9 is in a single-stranded state, a DNA-DNA double helix, and a DNA-RNA double helix when the excitation light of 510 nm is used.

Fig. 12: Fig. 12 shows the UV spectra of three samples when the fluorescent probe of another embodiment is in a single-strand state, DNA-DNA double helix and DNA-RNA double helix.

Fig. 13: Fig. 13 shows the fluorescence spectra of the three samples when the fluorescent probe in Fig. 12 is single-stranded, DNA-DNA double helix and DNA-RNA double helix.

Fig. 14: Fig. 14 shows the UV spectra of three samples when the fluorescent probe in another embodiment is in a single-stranded state, a DNA-DNA double helix, and a DNA-RNA double helix.

Fig. 15: Fig. 15 shows the fluorescence spectra of three samples when the fluorescent probe in Fig. 14 is in single-stranded state, DNA-DNA double helix and DNA-RNA double helix.

Fig. 16: Fig. 16 shows the UV spectra of three samples when the fluorescent probe of another embodiment is in a single-stranded state, when DNA-DNA double helix and DNA-RNA double helix.

Figure 17: Figure 17 shows the UV spectra of the three samples when the fluorescent probe in Figure 16 is in a single-stranded state, when the DNA-DNA double helix and when the DNA-RNA double helix.

Figure 18: Figure 18 is a graph showing the absorption, excitation and emission spectra of several fluorescent probes in the examples.

Fig. 19: Fig. 19 is a graph showing the absorption spectrum, excitation spectrum and emission spectrum of other fluorescent probes in Examples.

Figure 20: Figure 20 is the absorption spectrum obtained by measuring the absorption spectrum of the fluorescent probe of the embodiment at various temperatures and concentrations.

Figure 21: Figure 21 is the CD spectrum of the double strand obtained by hybridizing the fluorescent probe of the example.

Figure 22: Figure 22 is a graph showing the absorption spectrum, excitation spectrum and emission spectrum of other fluorescent probes in the examples.

Fig. 23: Fig. 23 is a diagram showing the fluorescence emission when other fluorescent probes of the examples are hybridized to obtain double strands.

Figure 24: Figure 24 is a graph showing the absorption and emission spectra of further fluorescent probes of the Examples.

Figure 25: Figure 25 is a graph showing the change in fluorescence when the RNA strand hybridized with the fluorescent probe of the example is digested by RNase H.

Fig. 26: Fig. 26 shows the observed changes in fluorescence intensity when changing the concentration ratio of the complementary DNA strand to the fluorescent probe of the example.

Fig. 27: Fig. 27 is a diagram showing the state of fluorescence emission in the imprint detection of the example.

Fig. 28: Fig. 28 is a photograph of differential interferometry when the fluorescent probe of the example is introduced into cells.

Fig. 29: Fig. 29 is a photo taken by observing fluorescence when the fluorescent probe of the example is introduced into cells.

Figure 30: Figure 30 is a photograph showing the overlay of Figures 28 and 29.

Fig. 31A: Fig. 31A is a photo taken by observing fluorescence when other fluorescent probes of the examples are introduced into cells.

Fig. 31B: Fig. 31B is a photo taken by observing fluorescence when some fluorescent probes of the example are introduced into cells.

Figure 32: Figure 32 is a graph showing the change in fluorescence over time after injecting the same probes as in Figures 28-30 into the nucleus.

Fig. 33: Fig. 33 is a photo taken by observing fluorescence when some other fluorescent probes of the examples are introduced into cells.

Detailed ways

Hereinafter, embodiments of the present invention will be described more specifically.

Compounds, nucleic acids and markers of the invention

The compounds and nucleic acids of the present invention are not particularly limited except that they are represented by the chemical formulas. As mentioned above, there is no particular limitation on its use, for example, it can be used as the marker of the present invention, or its synthetic raw material or synthetic intermediate. More detailed descriptions of the compounds, nucleic acids and markers of the present invention are as follows.

Among the compounds of the present invention, in said formulas (1), (1b) and (1c),

E is preferably, for example, an atomic group having a main chain structure of DNA, modified DNA, RNA, modified RNA, LNA or PNA (peptide nucleic acid).

Furthermore, in the formulas (1) and (1c),

[chem 27]

The atomic group shown is preferably an atomic group represented by any one of the following formulas (2)-(4),

[chem 28]

In the formula (1b),

[chem 29]

The atomic group shown is preferably an atomic group represented by any one of the following formulas (2b) to (4b).

[chem 30]

In said formula (2)-(4) and (2b)-(4b),

A is a hydrogen atom, a hydroxyl group, an alkyl group or an electron-withdrawing group,

M and J are each CH ₂ , NH, O or S, which may be the same or different,

B, X and Y are each the same as in said formula (1), (1b) or (1c),

In the formulas (2), (3), (2b) and (3b), more than one O atom in the phosphate bridge can be replaced by an S atom.

In view of easiness of synthesis, E is preferably an atomic group having a main chain structure of DNA, modified DNA, RNA, or modified RNA, for example, but may also be an atomic group having a main chain structure of LNA or PNA (peptide nucleic acid).

In the formulas (2) and (2b),

In A, preferably, for example, the alkyl group is a methoxy group, and the electron-withdrawing group is a halogen.

In said formula (1), (1b) or (1c),

Each of the main chain lengths (number of main chain atoms) of L ¹ , L ² and L ³ is preferably an integer of 2 or more. The upper limit of the main chain length (the number of main chain atoms) of L ¹ , L ² and L ³ is not particularly limited, and is, for example, 100 or less, more preferably 30 or less, particularly preferably 10 or less.

The compound of the present invention is preferably, for example, a compound represented by the following formula (5), (6), (6b) or (6c), a tautomer or stereoisomer thereof, or a salt thereof.

[chem 31]

In said formula (5), (6), (6b) and (6c),

l, m and n are arbitrary values, which can be the same or different,

B, E, Z ¹¹ , Z ¹² , X, Y and T are the same as in the formulas (1) and (1b).

In said formula (5), (6), (6b) and (6c),

Each of l, m and n is preferably an integer of 2 or more. The upper limits of l, m, and n are not particularly limited, and are, for example, 100 or less, more preferably 30 or less, particularly preferably 10 or less.

Among the compounds of the present invention, Z ¹¹ and Z ¹² are preferably atomic groups exhibiting an exciton effect. Thereby, for example, the increase in fluorescence increases when a double helix structure is formed, and the double helix structure can be detected more effectively. However, in the compound of the present invention, even if Z ¹¹ and Z ¹² are not atomic groups exhibiting excitonic effects, or only one atomic group (dye) exhibiting fluorescence is introduced into one molecule, the double helix can be effectively detected. structure.

Z ¹¹ and Z ¹² are, for example, preferably fluorescent atomic groups as described above. There is no particular limitation on the fluorescent atomic group. Z ¹¹ and Z ¹² are, for example, independently more preferably derived from thiazole orange, oxazole yellow, cyanine, hemicyanine, other cyanine dyes, methyl red, azo dyes or derivatives thereof group. In addition, groups derived from other known dyes are also suitable for use. Various fluorescent dyes that change the fluorescence intensity by binding to nucleic acids such as DNA have been reported. A typical example is that ethidium bromide is known to show strong fluorescence by intercalating into the DNA double helix structure, and is mostly used for DNA detection. In addition, pyrene carboxamide and prodan are also known as fluorescent dyes that can control the fluorescence intensity according to small polarity. In addition, the thiazole orange is a fluorescent dye in which a benzothiazole ring is linked to a quinoline through a methine group, and usually exhibits weak fluorescence, but generates strong fluorescence by inserting into DNA with a double helix structure. In addition, other dyes such as fluorescein yellow and Cy3 can be cited.

In addition, it is more preferable that Z ¹¹ and Z ¹² are, for example, each independently an atomic group represented by any one of the following formulas (7) to (9).

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[chem 33]

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In formula (7)-(9),

X ¹ and X ² are each S or O, which can be the same or different,

n is 0 or a positive integer,

R ¹ -R ¹⁰ , R ¹³ -R ²¹ are each independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group or an amino group,

Among R ¹¹ and R ¹² , one is L ¹ or L ² in the formula (1), (1b) or (1c), or the formula (5), (6), (6b) and (6c) In the linking group bonded to NH, the other is a hydrogen atom or a lower alkyl group,

Where multiple R ¹⁵ exist in formula (7), (8) or (9), they may be the same or different,

Where multiple R ¹⁶ exist in formula (7), (8) or (9), they may be the same or different,

X ¹ , X ² and R ¹ to R ²¹ in Z ¹¹ and X ¹ , X ² and R ¹ to R ²¹ in Z ¹² may be the same or different from each other.

In formula (7)-(9),

Among R ¹ -R ²¹ , more preferably, the lower alkyl group is a straight chain or branched chain alkyl group with 1-6 carbon atoms, and the lower alkoxy group is a straight chain or branched chain group with 1-6 carbon atoms. alkoxyl.

In formula (7)-(9),

In R ¹¹ and R ¹² , it is more preferable that the linking group is a polymethylene carbonyl group with 2 or more carbon atoms, through the carbonyl part and the ^L in the formula (1), (1b) or (1c) Or L ² , the NH bond in the formulas (5), (6), (6b) and (6c) described above. The upper limit of the number of carbon atoms of the polymethylenecarbonyl group is not particularly limited, and is, for example, 100 or less, preferably 50 or less, more preferably 30 or less, particularly preferably 10 or less.

When Z ¹¹ and Z ¹² are represented by the above-mentioned formulas (7) to (9), it is more preferable that they are each independently represented by, for example, a group represented by formula (19) or (20).

[chem 35]

[chem 36]

In formulas (19) and (20), X ¹ represents -S- or -O-. R ¹ -R ¹⁰ , R ¹³ and R ¹⁴ each independently represent a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group or an amino group. One of R ¹¹ and R ¹² represents L ¹ or L ² bonded to said formula (1), (1b) or (1c), said formula (5), (6), (6b) and ( The linking group on NH in 6c), the other in R ¹¹ and R ¹² represents a hydrogen atom or a lower alkyl group.

The compound of the present invention may be, for example, a compound having a structure represented by the following formula (10), a tautomer or a stereoisomer thereof, or a salt thereof.

[chem 37]

In formula (10),

E, Z ¹¹ , Z ¹² , Q, X and Y are the same as in the formula (1).

In the formulas (1), (1b) and (1c), B may have a natural nucleic acid base skeleton, but as mentioned above, it may also have an artificial nucleic acid base skeleton.

For example, B is preferably the structure shown in Py, Py der., Pu or Pu der.,

in,

The Py is an atomic group having a covalent bond with E at the 1-position and a covalent bond with the linker part at the 5-position in the six-membered ring represented by the following formula (11),

The Py der. is an atomic group obtained by replacing at least one of all the atoms of the six-membered ring of Py with an N, C, S or O atom, and the N, C, S or O atom may have a charge as appropriate , a hydrogen atom or a substituent,

The Pu is an atomic group having a covalent bond bonded to E at the 9-position in the condensed ring shown in the following formula (12), and having a covalent bond bonded to the linker part at the 8-position,

The Pu der. is an atomic group obtained by replacing at least one atom in the five-membered ring of Pu with an N, C, S or O atom, and the N, C, S or O atom may suitably have a charge, hydrogen atoms or substituents,

[chem 38]

The compound of the present invention may be, for example, a compound represented by the following formula (13) or (14), a tautomer or stereoisomer thereof, or a salt thereof.

[chem 39]

[chemical 40]

In the formulas (13) and (14), E, Z ¹¹ , Z ¹² , Q, X and Y are the same as in the formula (1). Py, Py der., Pu and Pu der. are as defined above.

When the compound of the present invention has a phosphoramidite group, the phosphoramidite group is preferably represented by the following formula (15), for example.

-P(OR ²² )N(R ²³ )(R ²⁴ ) (15)

In formula (15), R ²² is a protecting group for a phosphoric acid group, and R ²³ and R ²⁴ are alkyl or aryl.

More preferably, in the formula (15), R ¹⁵ is cyanoethyl, in R ¹⁶ and R ¹⁷ , the alkyl is isopropyl, and the aryl is phenyl.

Among the compounds of the present invention, for example, the compound represented by the formula (1) may be a compound represented by the following formula (21).

[chem 41]

In formula (21), A represents a hydrogen atom or a hydroxyl group. Preferably, A is a hydrogen atom. B represents a residue of adenine, guanine, cytosine, thymine or uracil. For example, adenine and guanine are bonded with a double bond at the 8-position, and cytosine, thymine, or uracil is bonded with a double bond at the 5-position. Z ¹¹ and Z ¹² each independently represent a fluorescent atomic group, a hydrogen atom or a protecting group for an amino group, particularly preferably a residue of a thiazole orange derivative or an oxazole yellow derivative. X represents a hydrogen atom, a hydroxy protecting group deprotectable with an acid, or a monophosphate, diphosphate or triphosphate group. Y is a hydrogen atom, a hydroxyl protecting group or a phosphoramidite group.

The compound represented by the formula (21) is more preferably a compound represented by the following formula (22).

[chem 42]

In formula (22), A represents a hydrogen atom or a hydroxyl group. Z ¹¹ and Z ¹² each independently represent an atomic group exhibiting fluorescence, a hydrogen atom or an amino protecting group, particularly preferably a residue of a thiazole orange derivative or an oxazole yellow derivative. X represents a hydrogen atom, a hydroxy protecting group deprotectable with an acid, or a monophosphate, diphosphate or triphosphate group. Y is a hydrogen atom, a hydroxyl protecting group or a phosphoramidite group.

In the compound of the formula (21) or (22), when Z ¹¹ and Z ¹² are hydrogen atoms or amino protecting groups, since there are two amino groups (or protected amino groups) in one molecule, the amino groups can be utilized Two molecules of labeling molecules are introduced into one molecule. For example, the sensitivity of nucleic acid detection can be improved by combining fluorescent substances, chemiluminescent substances, and the like to produce labeled nucleic acids. Furthermore, when Z ¹¹ and Z ¹² are atomic groups exhibiting fluorescence, nucleic acid detection can be easily performed by labeling with a specific fluorescent substance.

In addition, among the compounds of the formula (21) or (22), the compound in which Z ¹¹ and Z ¹² are atomic groups exhibiting fluorescence is modified with 2 molecules of fluorescent molecules such as thiazole orange derivatives or oxazole yellow derivatives of nucleotides. A probe formed from a single-stranded nucleic acid containing such a compound is quenched by exciton coupling, and the fluorescence is very weak in the state of the probe alone, but strong fluorescence can be exhibited by hybridization with DNA or RNA . That is, for example, the fluorescence of a thiazole orange derivative or an oxazole yellow derivative is strongly suppressed by such a twisted structure, but the twist of the structure is eliminated and fixed by binding the thiazole orange derivative or oxazole yellow derivative to DNA , showing strong fluorescence. Fluorescence can be detected by excitation with, for example, 488 nm, 514 nm Ar laser, but is not limited thereto.

The compound of the present invention represented by the formula (1), (1b) or (1c) can be used, for example, in the synthesis of nucleic acid (polynucleotide). That is, the compound of the present invention can be used as a nucleic acid labeling substance (nucleic acid labeling reagent). For example, by using the compound of the present invention represented by the formula (1), (1b) or (1c) as a nucleotide substrate, performing a nucleic acid synthesis reaction using a single-stranded nucleic acid as a template, or by using the formula ( 1), the compound of the present invention represented by (1b) or (1c) chemically synthesizes single-stranded nucleic acid (for example, chemical synthesis methods such as the phosphoramidite method using an automatic nucleic acid synthesizer), and can be produced to contain at least one molecule or more in one molecule. Nucleic acids of compounds of the invention. In this case, each of the atomic groups Z ¹¹ and Z ¹² may be an atomic group exhibiting fluorescence, or may be a hydrogen atom or a protecting group.

The structure of the nucleic acid of the present invention is as described above and contains at least one structure represented by the following formula (16), (16b), (17), (17b), (18) or (18b). In addition, the nucleic acids of the present invention may contain tautomers or stereoisomers of these structures, or salts thereof.

[chem 43]

[chem 44]

[chem 45]

[chem 46]

[chem 47]

[chem 48]

In formulas (16), (16b), (17), (17b), (18) and (18b),

B, E, Z ¹¹ , Z ¹² , L ¹ , L ² , L ³ , D and b are each a structure shown in the formula (1), (1b) or (1c),

In formulas (16), (17) and (18), E is the atomic group described in (i) in the formula (1), (1b) or (1c), and more than one O atom in the phosphate bridge can be replaced by S atom substitution,

In formulas (16b), (17b) and (18b), E is the atomic group described in (ii) in formula (1), (1b) or (1c).

In formulas (17) and (17b), each B may be the same or different, and each E may be the same or different.

In the formulas (16), (17), (16b), (17b), (18) and (18b),

Each of Z ¹¹ and Z ¹² is an atomic group exhibiting fluorescence, and may be the same or different.

The basic skeleton of the nucleic acid of the present invention is not particularly limited, and may be, for example, any of DNA, modified DNA, RNA, modified RNA, LNA, or PNA (peptide nucleic acid), or other structures. Furthermore, the number of bases in the nucleic acid of the present invention is not particularly limited, and is, for example, about 10 bp to 10 kb, preferably about 10 bp to 1 kb. Furthermore, when the nucleic acid of the present invention is an oligonucleotide, its length is not particularly limited, and is, for example, about 10-100 bp, more preferably about 10-50 bp, and still more preferably about 10-30 bp.

The number of compounds of formula (1), (1b) or (1c) contained in the nucleic acid of the present invention is not particularly limited, and may be about 1-100, preferably about 1-20.

The compound or nucleic acid of the present invention may have, for example, a structure represented by any one of the following formulas (23) to (25). Thus, it can be advantageously used as a dye-introduced fluorescent probe. However, compounds of the present invention suitable for use as fluorescent probes are not limited thereto.

[chem 49]

In the formula (23), two dyes (Fluo) are connected to the base B. The position where the base B is bonded to the linker is not particularly limited, for example, at one of the 4, 5 or 6 positions of pyrimidine, the 2, 3, 6, 7 or 8 positions of purine and the linker connect. The linker has one base linking site, which is divided into two or more branches along the way, and the end is linked with a dye. The method of linking with a base or a dye can use an amide bond, an ester bond, a disulfide bond, or a bond formed by a metal-catalyzed reaction for a double bond or a triple bond, a ring-forming condensation reaction, or a Michael addition reaction, etc. bonds, bonds generated by imine formation reactions, etc. For the linker, its length (l, m, n) can be selected freely, and can contain single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, amine, imine, ether bond, thioether bond, sulfur ester bond etc. In addition, it is preferable not to affect the excitonic effect caused by dimerization. Branches (X) are atoms of carbon, silicon, nitrogen, phosphorus, and boron, and may be protonated (eg, NH ⁺ ) or oxidized (eg, P=O). Preferably, the dye is a substance that exhibits an excitonic effect by dimerization, and the linker may be attached to any part of the dye. In formula (23), although deoxyribonucleic acid as the partial structure of DNA is shown, as an alternative, the nucleic acid backbone can also be 2'-O-methyl RNA or 2'-fluoro in addition to ribonucleic acid (RNA). Sugar-modified nucleic acids such as DNA, phosphorothioate-modified nucleic acids such as phosphorothioate nucleic acids, and functional nucleic acids such as PNA and LNA (BNA).

[chemical 50]

In the formula (24), two dyes (Fluo) are linked to the base B. The position where the base B is bonded to the linker is not particularly limited, for example, two positions in the 4, 5 or 6 positions of the pyrimidine, 2, 3, 6, 7 or 8 positions of the purine and the linker body connection. The two linkers each have a base attachment site and are attached to a dye at the other end. The method of linking with a base or a dye can use an amide bond, an ester bond, a disulfide bond, or a bond formed by a metal-catalyzed reaction for a double bond or a triple bond, a ring-forming condensation reaction, or a Michael addition reaction, etc. bonds, bonds generated by imine formation reactions, etc. For the linker, its length (l, m) can be selected freely, and can contain single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, amine, imine, ether bond, thioether bond, thioester bond wait. In addition, it is preferable not to affect the excitonic effect caused by dimerization. Preferably, the dye is a substance that exhibits an excitonic effect by dimerization, and the linker may be attached to any part of the dye. In formula (24), although deoxyribonucleic acid as the partial structure of DNA is shown, as an alternative, the nucleic acid backbone can also be 2'-O-methyl RNA or 2'-fluoro in addition to ribonucleic acid (RNA). Sugar-modified nucleic acids such as DNA, phosphorothioate-modified nucleic acids such as phosphorothioate nucleic acids, and functional nucleic acids such as PNA and LNA (BNA).

[Chemical 51]

In formula (25), one dye (Fluo) is linked to each base (B ¹ , B ² ) of consecutive nucleotides. There is no particular limitation on the position where each base is bonded to the linker, for example, at one of the 4, 5 or 6 positions of pyrimidine, the 2, 3, 6, 7 or 8 positions of purine and the linker body connection. The two linkers each have a base attachment site and are attached to a dye at the other end. The method of linking with a base or a dye can use an amide bond, an ester bond, a disulfide bond, or a bond formed by a metal-catalyzed reaction for a double bond or a triple bond, a ring-forming condensation reaction, or a Michael addition reaction, etc. bonds, bonds generated by imine formation reactions, etc. For the linker, its length (l, m) can be selected freely, and can contain single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, amine, imine, ether bond, thioether bond, thioester bond wait. In addition, it is preferable not to affect the excitonic effect caused by dimerization. It is preferable to use a dye that exhibits an excitonic effect by dimerization, and the linker may be attached to any part of the dye. In formula (25), although deoxyribonucleic acid as the partial structure of DNA is shown, as an alternative, the nucleic acid backbone can also be 2'-O-methyl RNA or 2'-fluoro in addition to ribonucleic acid (RNA). Sugar-modified nucleic acids such as DNA, phosphorothioate-modified nucleic acids such as phosphorothioate nucleic acids, and functional nucleic acids such as PNA and LNA (BNA).

In addition, in the case where there are isomers such as tautomers or stereoisomers (eg, geometric isomers, conformational isomers, and optical isomers) in the compound or nucleic acid of the present invention, any isomer may be used in the present invention. In addition, the salts of the compounds or nucleic acids of the present invention may be acid addition salts or base addition salts. In addition, the acid forming the acid addition salt may be an inorganic acid or an organic acid, and the base forming the base addition salt may be an inorganic base or an organic base. The inorganic acid is not particularly limited, and examples thereof include sulfuric acid, phosphoric acid, hydrofluoric acid, hydrochloric acid, hydrobromic acid, hydroiodic acid, hypofluorous acid, hypochlorous acid, hypobromous acid, hypoiodous acid, fluorous acid, Chlorous acid, bromous acid, iodic acid, fluoric acid, chloric acid, bromic acid, iodic acid, perfluoric acid, perchloric acid, perbromic acid and periodic acid, etc. The organic acid is not particularly limited, and examples thereof include p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromobenzenesulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, and acetic acid. The inorganic base is not particularly limited, and examples thereof include ammonium hydroxide, alkali metal hydroxides, alkaline earth metal hydroxides, carbonates, and bicarbonates, and more specifically, examples include sodium hydroxide, hydrogen Potassium oxide, potassium carbonate, sodium carbonate, sodium bicarbonate, potassium bicarbonate, calcium hydroxide and calcium carbonate, etc. The organic base is not particularly limited, and examples thereof include ethanolamine, triethylamine, and tris(hydroxymethyl)aminomethane. The method for producing the salt is also not particularly limited, for example, it can be produced by appropriately adding the acid or base to the electron donor/acceptor linking molecule by a known method. In addition, when there are isomers in the substituent or the like, any isomer may be used. For example, in the case of "naphthyl", it may be 1-naphthyl or 2-naphthyl.

In addition, in the present invention, the alkyl group is not particularly limited, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl, etc., may also be It is a group containing an alkyl group in the structure (alkylamino, alkoxy, etc.). In addition, the perfluoroalkyl group is not particularly limited, and examples include perfluoroalkyl groups derived from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl. group, or a group containing a perfluoroalkyl group in its structure (perfluoroalkylsulfonyl group, perfluoroacyl group, etc.). In the present invention, the acyl group is not particularly limited, and examples thereof include formyl, acetyl, propionyl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, cyclohexanoyl, benzoyl, ethoxy carbonyl, etc., or a group containing an acyl group in its structure (acyloxy, alkanoyloxy, etc.). In addition, in the present invention, the number of carbon atoms of the carbonyl group is included in the number of carbon atoms of the acyl group, for example, an alkanoyloxy group (acyl group) having 1 carbon atom means a formyl group. In addition, in the present invention, "halogen" refers to any halogen element, for example, fluorine, chlorine, bromine, and iodine are exemplified. In addition, in the present invention, the amino protecting group is not particularly limited, for example, trifluoroacetyl, formyl, C1-6 alkyl-carbonyl (such as acetyl, ethylcarbonyl, etc.), C1-6 alkyl- Sulfonyl, tert-butoxycarbonyl (hereinafter referred to as Boc), benzyloxycarbonyl, allyloxycarbonyl, fluorenylmethoxycarbonyl, arylcarbonyl (such as phenylcarbonyl, naphthylcarbonyl, etc.), aryl Sulfonyl (such as phenylsulfonyl, naphthylsulfonyl, etc.), C1-6 alkoxycarbonyl (such as methoxycarbonyl, ethoxycarbonyl, etc.), C7-10 aralkylcarbonyl (such as benzylcarbonyl, etc.) ), methyl, aralkyl (such as benzyl, diphenylmethyl, trityl, etc.), etc. The group can be substituted by 1 to 3 halogen atoms (such as fluorine, chlorine, bromine, etc.), nitro, etc., and its specific examples can include p-nitrobenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, m-chlorobenzyl Oxycarbonyl, p-methoxybenzyloxycarbonyl and the like. In addition, in the present invention, there is no particular limitation on the hydroxyl protecting group (including substances that can be deprotected with an acid), and examples include dimethoxytrityl, monomethoxytrityl, 9- (9-phenyl)xanthenyl (pixyl) and the like.

As the compound or nucleic acid of the present invention, particularly preferred are compounds or nucleic acids such as those described in Examples below, particularly compounds (nucleic acids) 102-106, 110, 113, 114, 116-118, 120, 121, 122, 123, 124, ODN1, ODN2, ODN3, ODN4, ODN5, ODN6, ODN7, ODN8, ODN9, ODN10, ODN(anti4.5S) and ODN(antiB1) and geometric isomers, stereoisomers and salts of these substances . In particular, compounds 110, 113, 114, 116-118, 120, 121, 122, 123, 124, ODN1, ODN2, ODN3, ODN4, ODN5, ODN6, ODN7, ODN8, ODN9, ODN10, ODN (anti4.5S) And ODN (antiB1) is particularly excellent in nucleic acid detection sensitivity because thiazole orange and DNA are covalently bonded through a unique structure. Also, compounds 110, 113, 117, 118, 120, 121, 122, 123, 124, ODN1, ODN2, ODN3, ODN4, ODN5, ODN9, ODN (anti4.5S) and ODN (antiB1) can be more effectively used as a fluorescent probe for single-stranded DNA that suppresses fluorescence in a single-stranded state and increases fluorescence intensity by forming a double helix with complementary DNA or RNA.

Next, the markers of the present invention are as described above,

(i) Markers: two planar chemical structures in a molecule are not in the same plane, but exist at an angle, but when the molecule is inserted or grooved into a nucleic acid, the two planar chemical structures Arranged in the same plane to produce fluorescent light;

(ii) A label formed of two or more groups of dye molecules in which, although the excitonic effect due to parallel aggregation of two or more dye molecules does not exhibit fluorescence emission, these molecules are bound to nucleic acid at insertion or groove During the middle, the release of the aggregation state will produce fluorescence emission; or

(iii) A complex marker characterized by a chemical structure having two or more dye molecules in the same molecule, wherein although the excitonic effect due to the parallel aggregation of two or more dye molecules is not Fluorescence is exhibited, but upon intercalation or groove binding of these molecules into a nucleic acid, fluorescence occurs upon release of the aggregated state.

In the case of (ii) or (iii) above, the dye molecule is preferably the molecule described in (i) above. In addition, in the case of (iii), it is preferable to have a structure in which two or more dye molecules are bonded to a linker molecule that is bonded to a nucleic acid to be labeled, and the dye molecule is bonded to the linker molecule by means of Additional linker molecules to form branched structures, or direct bonding without the aid of additional linker molecules.

The label of the present invention is preferably the compound of the present invention wherein the atomic groups ^Z11 and ^Z12 are atomic groups exhibiting fluorescence, tautomers or stereoisomers thereof, or salts thereof, or wherein ^Z11 and Z ¹² is said nucleic acid of the present invention, a tautomer or stereoisomer thereof, or a salt thereof, which is an atomic group exhibiting fluorescence. For example, in the compound or nucleic acid of the present invention, since Z ¹¹ and Z ¹² are atomic groups exhibiting an excitonic effect, the increase in fluorescence when a double helix structure is formed becomes larger, and the double helix structure can be detected more efficiently. However, in the compound or nucleic acid of the present invention, even if ^Z11 and ^Z12 are not atomic groups exhibiting an excitonic effect, or only one atomic group (dye) exhibiting fluorescence is introduced into one molecule, it can still be used as an atomic group (dye) for nucleic acid, etc. Marker, effectively detect the double helix structure. The form of the label of the present invention is, for example, a form of a fluorescent probe for single-stranded nucleic acid, but is not limited thereto, and any form such as labeled mononucleotide, labeled oligonucleotide, or double-stranded nucleic acid may be used.

In addition, the markers of the present invention, for example,

For labeling mononucleotides, labeling oligonucleotides, labeling nucleic acids or labeling nucleic acid analogues,

Wherein the label is the label described in any one of (i)-(iii), or the compound of the present invention in which Z ¹¹ and Z ¹² are atomic groups exhibiting fluorescence, and tautomers thereof or stereoisomers, or salts thereof, or the nucleic acid of the present invention wherein Z ¹¹ and Z ¹² are atomic groups exhibiting fluorescence, tautomers or stereoisomers thereof, or salts thereof are labeled.

Alternatively, the markers of the present invention, for example,

For labeling mononucleotides, labeling oligonucleotides, labeling nucleic acids or labeling nucleic acid analogues,

Wherein the label is by means of a linker molecule bonded to one or more base molecules or main chain constituent molecules in a single nucleotide, oligonucleotide, nucleic acid or nucleic acid analogue, the (i )-(iii), or the compound of the present invention in which Z ¹¹ and Z ¹² are atomic groups exhibiting fluorescence, tautomers or stereoisomers thereof, or salts thereof , or Z ¹¹ and Z ¹² are labeled with the nucleic acid of the present invention, its tautomer or stereoisomer, or a salt thereof, which are atomic groups exhibiting fluorescence.

Alternatively, markers of the present invention such as

For labeling mononucleotides, labeling oligonucleotides, labeling nucleic acids or labeling nucleic acid analogues,

Wherein the label is bonded to the 5-position carbon atom of the pyrimidine nucleus or the 8-position carbon atom of the purine nucleus of one or more base molecules in a single nucleotide, oligonucleotide, nucleic acid or nucleic acid analog A linker molecule, the compound of the present invention in which the label described in any one of (i)-(iii), or Z ¹¹ and Z ¹² is an atomic group exhibiting fluorescence, or its tautomorphism isomer or stereoisomer, or a salt thereof, or Z ¹¹ and Z ¹² are atomic groups exhibiting fluorescence, the nucleic acid of the present invention, its tautomer or stereoisomer, or a salt thereof is labeled.

Compounds of the present invention and methods for producing nucleic acids

The method for producing the compound and nucleic acid of the present invention is not particularly limited, and known synthesis methods (production methods) can be suitably used. For example, in the case of the compound represented by the formula (21), it can be produced by a production method comprising the following steps: after activating the carboxyl group of the compound represented by the following formula (26), it is combined with tris(2-aminoethyl group) amine reaction; protection of amino group; and the reaction of protecting the hydroxyl group present in the compound obtained above with a protecting group, and the reaction of adding a phosphoric acid or phosphoramidite group to the hydroxyl group present in the obtained compound.

[Chemical 52]

In formula (26), A represents a hydrogen atom or a hydroxyl group. B represents a residue of adenine, guanine, cytosine, thymine or uracil.

The production method (synthesis method) usable for the production of the compound or nucleic acid of the present invention is, for example, the following. That is, first, as a method of simply labeling DNA, a method of reacting an active amino group in DNA with an activated carboxyl group in a labeling agent in a buffer solution is widely used. This method can also be used in the manufacture of any of the compounds or nucleic acids of the invention, particularly for the introduction of linkers or dyes. The amino group introduction method may be a method using Amino modifier phosphoramidite sold by GLEN RESEARCH.

The atomic groups Z ¹¹ and Z ¹² can, for example, be changed from protecting groups to hydrogen atoms (removal of protecting groups), and then the hydrogen atoms can be replaced with fluorescent atomic groups (dye). The method for removing the protecting group is not particularly limited, and known methods can be used appropriately. There is no particular limitation on the method of substitution with a fluorescent atomic group (dye). For example, the compound or nucleic acid of the present invention in which Z ¹¹ and Z ¹² are hydrogen atoms can be appropriately reacted with a fluorescent molecule (dye). For example, when at least one of Z ¹¹ and Z ¹² is an active amino group, it is preferable because it easily reacts with a fluorescent molecule (dye), and it is more preferable that both of Z ¹¹ and Z ¹² are active amino groups. Fluorescent molecules (dye) are not particularly limited, and can be, for example, compounds shown in any one of the formulas (7)-(9) (wherein, any one of R ^{and R} can be a hydrogen atom Or lower alkyl or carboxypolymethylene). In addition, in the case of nucleic acids (polynucleotides, polynucleosides, oligonucleotides, or oligonucleotides), the step of removing the protective group and replacing it with a fluorescent atomic group (dye) can be performed during polymerization (nucleic acid synthesis) ) before, or after. For example, it is preferable to introduce a fluorescent atomic group (dye) after polymerization (nucleic acid synthesis) in view of preventing damage to the dye portion during the synthesis step.

The dyes are not particularly limited as described above, and all dyes can be used, but preferred are, for example, cyanine dyes, and thiazole orange is particularly preferred. A cyanine dye is, for example, a chemical structure in which two heterocyclic rings having heteroatoms are linked by a methine linker. Fluorescent dyes with various excitation/emission wavelengths can be synthesized by changing the type of heterocycle and the length of the methine linker, or introducing substituents on the heterocycle, etc. In addition, introduction of a linker for introduction of DNA is relatively easy. In addition, thiazole orange does not fluoresce substantially in water, but fluoresces strongly by interacting with DNA or RNA. It is believed that the interaction between dye molecules is inhibited through the interaction with nucleic acids, which in turn inhibits the rotation of the methine linker between the two heterocycles surrounding the dye molecule, resulting in an increase in fluorescence intensity. In addition, the method of using thiazole orange dye is well known, and can be used with reference to, for example, the following documents: H.S.Rye, M.A.Quesada, K.Peck, R.A.Mathies and A.N.Glazer, High-sensitivity two-color detection of double-stranded DNA with a Confocal fluorescence gel scanner using ethidium homodimer and thiazole orange, Nucleic Acids Res., 1991, 19, 327-33; and L.G.Lee, C.H.Chen and L.A. Chiu, Thiazole orange: a newdye for reticulocyte analysis, 1, 59 Cytometry, 7 17.

The basic skeleton of the compound or nucleic acid of the present invention is not particularly limited as described above, and may be, for example, any of DNA, modified DNA, RNA, modified RNA, LNA, or PNA (peptide nucleic acid), or other structures. When DNA, modified DNA, RNA, or modified RNA is used as the basic skeleton, synthesis is easy, and substitution with dye (introduction of dye molecules) is also easy, so it is preferable. The method for introducing dye molecules into LNA or PNA is not particularly limited, and known methods can be suitably used. Specifically, for example, the following documents can be referred to: Analytical Biochemistry 2000, 281, 26-35. Svanvik, N., Westman, G., Wang, D., Kubista, M. (2000) Anal Biochem.281, 26-35 Hrdlicka, P.J., Babu, B.R., Sorensen, M.D., Harrit, N., Wengel, J. (2005) J. Am. Chem. Soc. 127, 13293-13299.

Methods for synthesizing nucleic acids having DNA, modified DNA, RNA, or modified RNA as their basic backbone are well known, for example, by the so-called phosphoramidite method. The phosphoramidite reagent as its raw material can also be easily synthesized by a known method. When the nucleic acid of the present invention is DNA, especially short oligomeric DNA, it can be easily synthesized using, for example, an automatic DNA synthesizer. In addition, long-chain nucleic acid (DNA) or the like can also be synthesized, for example, by PCR or the like. The bonding position of DNA and the dye molecule is not particularly limited as described above, but the 5th position of thymidine is particularly preferable. It is known that triphosphates of nucleotide derivatives protruding from the 5-position of thymidine with various substituents are introduced with high efficiency by DNA polymerase. Therefore, for example, the nucleic acid of the present invention can be easily synthesized not only when it is a short oligomeric DNA, but also when it is a long-chain DNA.

In particular, the use of single-stranded DNA such as thiazole orange as the fluorescent probe (label) of the present invention has advantages such as the following: (1) It can be synthesized only by adding a dye to DNA synthesized by an automatic DNA synthesizer in a buffer solution (2) By reacting the long-chain DNA prepared by the enzymatic method with the dye, the long-chain fluorescent probe can be prepared. Alternatively, excitation can be performed with light having a longer wavelength, for example, around 500 nm.

Nucleic acid detection methods and kits

As described above, the nucleic acid detection method of the present invention is

(1) nucleic acid detection method comprising the following steps:

Using the label of the present invention—labeled mononucleotide or labeled oligonucleotide as a substrate to carry out nucleic acid synthesis, thereby synthesizing a double-stranded nucleic acid inserted or groove-bonded with the fluorescent atomic group or dye molecular structure;

separately measuring the fluorescence intensity before and after the double-stranded nucleic acid synthesis step; and

Detecting nucleic acid synthesis by comparing the fluorescence intensity before and after the double-stranded nucleic acid synthesis step;

(II) A nucleic acid detection method comprising the following steps:

Using the marker of the present invention—single-stranded nucleic acid as the first nucleic acid, hybridizing it with a second nucleic acid having a sequence complementary to the first nucleic acid or a sequence similar to the complementary sequence to perform nucleic acid synthesis, thereby synthesizing the insertion A double-stranded nucleic acid with or groove-bonded with the fluorescent atomic group or dye molecular structure;

separately measuring the fluorescence intensity before and after the double-stranded nucleic acid synthesis step; and

Detecting hybridization between the first nucleic acid and the second nucleic acid by comparing the fluorescence intensity before and after the double-stranded nucleic acid synthesis step;

(III) A nucleic acid detection method comprising the following steps:

Using the marker of the present invention—single-stranded nucleic acid as the first nucleic acid, hybridizing it with a second nucleic acid having a sequence complementary to the first nucleic acid or a sequence similar to the complementary sequence to perform nucleic acid synthesis, thereby synthesizing the insertion A double-stranded nucleic acid with or groove-bonded with the fluorescent atomic group or dye molecular structure;

separately measuring the fluorescence intensity before and after the double-stranded nucleic acid synthesis step; and

Detecting hybridization between the first nucleic acid and the second nucleic acid by comparing the fluorescence intensity before and after the double-stranded nucleic acid synthesis step;

or

(IV) A nucleic acid detection method, characterized in that a third nucleic acid is used to detect the formation of a triple-stranded nucleic acid or a nucleic acid analogue, the third nucleic acid having the first nucleic acid sequence, the second nucleic acid sequence, or a combination of these sequences Complementary sequences, or sequences similar to said sequences complementary to these sequences, are labeled or not labeled with the markers or complex markers of the present invention.

Preferably, (a) more than 2 dye molecules are bonded to one base molecule in the first nucleic acid through a linker, and (b) more than 2 dye molecules are bonded to One base molecule in the first nucleic acid, or (c) two or more dye molecules are bonded to two adjacent base molecules in the first nucleic acid via one or more linkers.

Said nucleic acid synthesis is preferably carried out eg enzymatically, but can also be carried out by other methods. In addition, the nucleic acid detection method of the present invention preferably uses the compound of the present invention whose Z ¹¹ and Z ¹² are atomic groups exhibiting fluorescence, its tautomer or stereoisomer, or a salt thereof, or has the A labeled nucleic acid that describes a part of the structure of the nucleic acid of the present invention detects double-stranded or triple-stranded nucleic acid.

Secondly, as mentioned above, the kit of the present invention comprises a nucleic acid synthesis device, a marker and a fluorescence intensity measurement device, wherein the marker is the marker of the present invention. That is, the kit of the present invention can detect nucleic acid with high sensitivity by using the marker of the present invention as the marker. Other than that, there is no particular limitation on the kit of the present invention. For example, the nucleic acid synthesis device is not particularly limited, and it may be, for example, a known automatic nucleic acid synthesizer or the like. In addition, there is no particular limitation on the fluorescence intensity measuring device, for example, a known fluorescence measuring instrument or the like can be used.

The kit of the present invention is preferably used in the nucleic acid detection method of the present invention, but is not limited thereto, and can be used for any purpose. In addition, the kit of the present invention is preferably used, for example, as a kit for research, clinical use or diagnosis.

Hereinafter, the nucleic acid detection method or kit of the present invention will be described in more detail. However, the nucleic acid detection method and kit of the present invention are not limited to the following description.

The marker of the present invention is used in the nucleic acid detection method of the present invention as described above. In this case, the labeling substance of the present invention may have only one but preferably two or more fluorescent atomic groups (dye) per molecule. Accordingly, for example, the fluorescent atomic group (dye) has an excitonic effect. By the excitonic effect, for example, the fluorescence intensity in the single-chain state can be suppressed, so that the double-helix structure can be detected more effectively. In addition, the so-called exciton effect (exciton coupling) means, for example, an effect in which a plurality of dyes aggregate in parallel to form H-aggregate (H-aggregate), and hardly emit fluorescence. This effect is thought to be due to the fact that the excited state of the dye is split into two energy levels by Davydov splitting, excitation to the higher energy level → internal conversion to the lower energy level → Luminescence is inhibited thermodynamically. However, the present invention is not limited to the above description. The occurrence of the excitonic effect can be confirmed based on the fact that the absorption band of the dye forming the H aggregate appears at a wavelength shorter than the absorption band of a single dye. Examples of dyes exhibiting this effect include the aforementioned thiazole orange and its derivatives, oxazole yellow and its derivatives, cyanine and its derivatives, hemicyanine and its derivatives, methyl red and its derivatives , other dyes commonly known as cyanine dyes, azo dyes.

These dyes readily form with DNA or RNA by intercalative bonding to DNA-DNA duplexes or DNA-RNA duplexes forming double helices, or to artificial nucleic acids such as phosphorothioate nucleic acids, PNA (peptide nucleic acid) or LNA (BNA). double chain. When a plurality of such dyes are introduced into the probe, the usual single-stranded state (that is, the state of only the probe before hybridization) is strongly quenched due to the excitonic effect, but when hybridized to the target DNA or RNA, the aggregate is released, The individual dyes are inserted into the duplex one after another. At this time, since there is no electronic interaction between the dyes, there is no excitonic effect and strong fluorescence is emitted. At this time, the absorption band of the dye is the same as that of a single dye, showing that there is no excitonic effect between the dyes. In addition, when the dye is intercalated into the double strand, the structural twist originally possessed by the dye is eliminated, and the fluorescent light becomes stronger.

Thus, for example, by designing probes that exhibit excitonic effects due to multiple dyes, it is possible to turn fluorescence on and off very clearly by hybridization to the target sequence. In addition, when only one molecule of dye is bound to the probe sequence, no excitonic effect is exhibited. For example, stronger fluorescence than that of single strands can be generated due to the flattening of the structure of the dye due to the intercalation of the dye due to the formation of double strands. Also, even if two or more molecules of dyes are bonded, the excitonic effect does not appear when the dyes are separated from each other by a distance at which electrons do not interact with each other. That is, in order to exhibit the excitonic effect, two or more molecules of the dye must be bonded to the compound of the present invention or the molecule of the nucleic acid in such a manner that the dyes are placed at a sufficiently close distance. That is, when the compound or nucleic acid of the present invention is used as a fluorescent probe, it is preferable to bind two or more dyes to one nucleotide in the probe, or to bind one dye to every two or more consecutive nucleotides. .

The nucleic acid detection method of the present invention can be schematically shown by, for example, FIG. 1 . The figure (A) (the left picture is the picture of "non-heterozygote") shows the probe quenched by the exciton effect, and the picture (B) (the right picture is the picture of "heterozygote") shows the probe quenched by the double The strand forms a probe that intercalates and fluoresces. In the figure, symbol 1 represents the nucleic acid (fluorescent probe) of the present invention. 2 represents an atomic group (dye) exhibiting fluorescence. 1' represents the complementary strand of nucleic acid (fluorescent probe) 1. 3 represents a double-stranded nucleic acid formed by 1 and 1'. Also, the upper part of the figure is the electron transfer map. "Allow" means transfer is permitted. "Prohibited" means that diversion is prohibited. "Emittable" means capable of fluorescing. "Non-emitting" means theoretically unable to fluoresce. That is, it is considered that in the single-chain state (FIG. 1(A)), the dye 2 in the base state aggregates to interact according to the exciton coupling theory, and the excited state of the dye aggregate is separated into two energy levels, thereby inhibiting glow. Since emission from lower energy levels is theoretically forbidden, the singlet excited state of the aggregates remains in the low emission state. On the other hand, it is considered that when a double strand is formed by hybridization ( FIG. 1(B) ), the dye 2 intercalates or grooves into the double-stranded nucleic acid 3 to eliminate exciton coupling, thereby generating fluorescence. However, FIG. 1 is a schematic diagram schematically showing an example of the nucleic acid detection mechanism of the present invention, and the present invention is not limited to FIG. 1 and the above description. In the exciton effect, fluorescence emission can be controlled by controlling the distance between two dyes. By associating this system with DNA for sequence discrimination, sequence-selective fluorescence can be obtained. In the nucleic acid detection method or kit of the present invention, for example, hybridization can be detected by irradiating the sample with visible light from below the sample, and it can also be clearly determined by visual inspection. In addition, in the nucleic acid detection method or kit of the present invention, hybridization can be observed in containers such as fluorescent chambers, microplates, gels, capillaries, and plastic tubes. Furthermore, in the nucleic acid detection method or kit of the present invention, for example, hybridization can be observed immediately after mixing with the target nucleic acid.

With the label, nucleic acid detection method or kit of the present invention, it is easy to perform fluorescent detection of sequence-specific nucleic acid even in environments where washing is difficult, such as real-time PCR or intracellular. More specifically, applications such as the following (1)-(7) can be performed. In addition, hereinafter, "the probe of the present invention" refers to a fluorescent probe that is one of the above-mentioned labels of the present invention. As mentioned above, the markers of the present invention are used in the nucleic acid detection methods and kits of the present invention. In addition, the following (1)-(7) are examples, and the marker, nucleic acid detection method or kit of the present invention is not limited to these descriptions.

(1) The probe of the present invention can be used for liquid-phase homogeneous detection (using 96-well microplate or capillary, etc.).

(2) The probe of the present invention can be used as a PCR probe. It can be used as an inexpensive method to detect amplification curves in DNA amplification reactions (real-time PCR) and replace TaqMan probes. Can be used as primer labels or internally labeled probes.

(3) The probes of the present invention can be used as capture probes or labeling probes in DNA chips. It is a high-throughput system that does not require reagents, and does not require labeling or washing. Human errors can be largely avoided. Multiple (high-throughput) analyzes can be performed simultaneously on glass or alternative solid-phase support materials (substrates such as gold, ITO, copper, etc., materials such as diamond or plastic to which multiple samples can be attached).

(4) The probe of the present invention can be immobilized on beads, fibers or hydrogels. Genes can be detected in semi-liquid/semi-solid environments. While having a liquid detection environment, it can also be delivered in solid form.

(5) The probe of the present invention can be used as a probe for blotting (Southern blotting, Northern blotting, dot blot, etc.). Detection can be performed by making only target gene fragments emit light. With the method of the present invention, no washing is required after hybridization.

(6) The probe of the present invention can be used as a probe for detection and tracking of nucleic acid in the nucleus. Thus, spatiotemporal analysis of intracellular DNA/RNA can be performed. A fluorescence microscope or cell sorter can be used. It can be applied to DNA labeling, tracking of RNA transcription/splicing, functional analysis of RNAi, etc. Washing is not required in the method of the present invention and is therefore suitable for tracking the function of living cells.

(7) The probe of the present invention can be used as a probe for fluorescence in situ hybridization (FISH). By the method of the present invention, tissue staining and the like can be performed. Washing is not required in the method of the present invention, so the error caused by man is small. That is, the probe of the present invention can be used as a fluorescent dye that does not fluoresce when a target biomolecule is not recognized, and thus a bioimaging process that does not require complicated washing steps can be established when using the probe. This enables real-time fluorescence observation with high reliability and low workload.

In addition, the effects of the fluorescent probe (label) of the present invention include, for example, the following advantages compared with conventional single-strand state quenched fluorescent probes (molecular beacons, etc.). However, these are also exemplary, and the present invention is not limited thereto.

(1) Synthesis is easy when only one dye is used.

(2) When the DNA probe (label) of the present invention has a free end, it can be easily used as a PCR probe.

(3) There is no need to form a special high-level structure such as a hairpin structure, so a sequence such as a stem sequence that is not related to sequence recognition is not required (there is no useless sequence, and there is no restriction on the sequence).

(4) Fluorescent dyes can be introduced at multiple positions (desired positions) of the probe.

(5) When two or more dye structures are included in one molecule, the positional relationship between the dyes is limited, so the S/N ratio (the ratio of fluorescence intensities before and after hybridization) is large.

The fluorescence intensity of the probes of the invention can be efficiently varied, for example, by controlling the exciton interactions of the bound dye moieties. In the present invention, in particular, by using the method of exciton interaction, since it can function as an on-off probe, sufficiently high quenching performance can be obtained. A number of distinct advantages are obtained compared to detection. The design of such on-off fluorescent nucleotides is very important for establishing biological imaging methods that do not require eg washing. The photophysical properties exhibited by probes utilizing the excitonic effect are not only very characteristic, but are also suitable for new types of fluorescence used in DNA sequencing (sequence determination), genotyping (genotype analysis), monitoring of DNA structural transitions, and observation of gene expression DNA probe design.

In addition, if the probe (nucleic acid) of the present invention is used, by quantifying, for example, the target nucleic acid sequence, the occurrence of phenomena such as amplification, decomposition, and protein bonding of the sequence can be detected immediately, and the amount of these phenomena can be quantified Quantify. The detection and quantification can be explained below, but these explanations are only exemplary and do not limit the present invention. That is, first, the probe (nucleic acid) of the present invention hybridizes with the target nucleic acid sequence at a constant mass ratio to form a double strand. The substance amount of the formed double strand is directly proportional to the substance amount of the target nucleic acid sequence, so by measuring the fluorescence intensity of the double strand, the target nucleic acid sequence can be detected and its substance amount can be quantified at the same time. In this case, since the fluorescence emission of the probe (nucleic acid) of the present invention is suppressed, the measurement of the fluorescence intensity of the double strands can be accurately measured without interfering with it.

Example

The present invention will be described more specifically by the following examples, but the present invention is not limited to the following examples. In addition, "ODN" hereinafter means oligomeric DNA (DNA oligomer).

Measurement conditions, etc.

Conventional commercially available reagents and solvents are used. The N-hydroxysuccinimide ester of biotin used was produced by PIERCE. As the silica gel used for compound purification, Wako gel C-200 (Wako Pure Chemical Industries, Ltd.) was used. ¹ H, ¹³ C and ³¹ P NMR spectra were measured by JNM-α400 (trade name) of JEOL (Japan Electronics Co., Ltd.). Coupling constants (J values) are expressed in Hertz (Hz). Chemical shifts are expressed in ppm. Dimethylsulfoxide (δ=2.48 in ¹ HNMR, δ=39.5 in ¹³ CNMR) and methanol (δ=3.30 in ¹ HNMR, δ=49.0 in ¹³ CNMR) were used as internal standards. In the measurement of ³¹ P NMR, H ₃ PO ₄ (δ=0.00) was used as an external standard. The ESI mass spectrum was measured using Bruker Daltonics APEC-II (trade name) of Bruker Corporation. As the automatic DNA synthesizer, 392DNA/RNA synthesizer (trade name) of Applied Biosystem was used. Reverse-phase HPLC uses the device Gilson Chromatograph of Gilson Company, Model 305 (commodity name) and Chemco Company's CHEMCOBOND 5-ODS-H preparative chromatographic column (commodity name, 10 * 150mm) to separate, through UV detector Model 118 (commercial name) name) was detected at a wavelength of 260 nm. DNA quality was determined by MALDI-TOF MS. MALDI-TOF MS uses the PerSeptive VoyagerElite (trade name) of Applied Biosystems Company, under accelerating voltage 21kV, under negative charge condition, uses 2 ', 3 ', 4'-trihydroxyacetophenone as matrix, uses T8 ([MH ].2370.61) and T17 ([MH].5108.37) as internal standards. The UV and fluorescence spectra were respectively measured using a Shimadzu UV-2550 (trade name) spectrophotometer and RF-5300PC (trade name) spectrofluorometer of Shimadzu Corporation. The fluorescence lifetime was measured with a small-sized high-performance fluorescence lifetime measuring instrument HORIBA JOBIN YVO N FluoroCube (trade name) of Horiba Manufacturing Co., Ltd. equipped with NanoLED-05A (trade name). Determination of the melting point (Tm) of double-stranded nucleic acids was performed at a final double-strand concentration of 2.5 μM in 50 mM sodium phosphate buffer (pH=7.0) containing 100 mM sodium chloride. The absorbance of the sample was measured at a wavelength of 260 nm, and was tracked while heating at a speed of 0.5 °C/min in the range of 10 °C to 90 °C. From the properties thus observed, the temperature at which the change first occurred was defined as the melting point Tm.

Unless otherwise specified, the determination of absorption spectrum, fluorescence spectrum and CD spectrum is at a chain concentration (single or double chain) of 2.5 μM in 50 mM sodium phosphate buffer (pH=7.0) containing 100 mM sodium chloride, Performed using a measuring cell with an optical path length of 1 cm. The bandwidth of excitation and fluorescence emission is 1.5nm. Using 9,10-diphenylanthracene as a reference substance, the fluorescence quantum yield (Φ _F ) was calculated based on the quantum yield Φ _F =0.95 of 9,10-diphenylanthracene in ethanol. The emission spectral area was calculated by integration using the device software. The quantum yield (Φ _F ) was calculated by the following formula (1).

Φ _F(S) /Φ _F(R) ＝[A _(S) /A _(R) ]×[(Abs) _(R) /(Abs) _(S) ]×[n _(S) ² /n _{(R )} ² ] (1)

In the above formula (1), Φ _F(S) is the fluorescence quantum yield of the sample, and Φ _F(R) is the fluorescence quantum yield of the control substance. A _(S) is the area of the fluorescence spectrum of the sample, and A _(R) is the area of the fluorescence spectrum of the control substance. (Abs) _(S) is the optical density of the sample solution at the excitation wavelength, and (Abs) _(R) is the optical density of the reference substance solution at the excitation wavelength. n _(S) is the refractive index of the sample solution, n _(R) is the refractive index of the control substance solution, calculated as n _(S) =1.333, n _(R) =1.383.

Example 1-3

According to the following scheme 1, compounds 102 and 103 in which two active amino groups were each protected with a trifluoroacetyl group were synthesized (produced), and phosphoramidite 104 was further synthesized.

[Chemical 53]

plan 1

Reagents and reaction conditions (a) (i) N-hydroxysuccinimide, EDC/DMF, (ii) tris(2-aminoethyl)-amine/CH ₃ CN, (iii) CF ₃ COOEt, Et ₃ N; (b) DMTrCl/pyridine; (c) 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite, 1H-tetrazole/ _CH3CN .

Regarding Scheme 1 above, it is described in more detail below.

Example 1: 2-[2-[N, N-bis(2-trifluoroacetylaminoethyl)]-aminoethyl]carbamoyl-(E)-vinyl]-2'-deoxyuridine ( Synthesis of 2-[2-[N, N-bis(2-trifluoroacetamidoethyl)]-aminoethyl]carbamoyl-(E)-vinyl)-2′-deoxyuridine, compound 102)

Starting material (E)-5-(2-carboxyvinyl)-2'-deoxyuridine ((E)-5-(2-carboxyvinyl)-2'-deoxyuridine, compound 101) according to Tetrahedron1987, 43, 20 , 4601-4607 Synthesis. That is, first, 71 mL of 1,4-dioxane was added to 430 mg of palladium(II) acetate (FW224.51) and 1.05 g of triphenylphosphine (FW262.29), and then 7.1 mL of triethylamine (FW 101.19, d=0.726), heated and stirred at 70°C. When the reaction solution changed from reddish brown to dark brown, add 14.2g 2'-deoxy-5-iodouridine (FW354.10) and 7.0mL methyl acrylate (FW 86.09, d=0.956) in 1,4-diox The suspension in alkanes was heated to reflux at 125°C for 1 hour. Afterwards, it was filtered while still hot, the residue was washed with methanol, and the filtrate was recovered. The solvent of the filtrate was distilled off under reduced pressure, and the product was purified with a silica gel column (5-10% methanol/dichloromethane). The solvent in the collected fractions was distilled off under reduced pressure, and the remaining white solid was dried under reduced pressure. About 100 mL of ultrapure water was added to this dried solid, 3.21 g of sodium hydroxide (FW40.00) was added, and it stirred overnight at 25 degreeC. Then, concentrated hydrochloric acid was added to acidify the solution, and the resulting precipitate was filtered off, washed with ultrapure water, and dried under reduced pressure. Thus, 8.10 g (yield 68%) of the target compound (compound 101) were obtained as a white powder. In addition, the white powder was confirmed to be the target compound 101 by the fact that the measured value by ¹ H NMR coincided with the reference value. In addition, ¹³ C NMR measured values are described below.

(E)-5-(2-carboxyvinyl)-2'-deoxyuridine (compound 101):

¹³ CNMR (DMSO-d6): δ168.1, 161.8, 149.3, 143.5, 137.5, 117.8, 108.4, 87.6, 84.8, 69.7, 60.8, 40.1.

Then, 1.20g (E)-5-(2-carboxyvinyl)-2'-deoxyuridine 101 (molecular weight 298.25), 925mg N-hydroxysuccinimide (molecular weight 115.09) and 1.54g 1-ethyl - 3-(3-Dimethylaminopropyl) carbodiimide (molecular weight: 191.70) was placed in a recovery flask (Nasuflasco) with a stirring bar, 20 mL of DMF was added, and the mixture was stirred at 25° C. for 16 hours. Add about 1 mL of acetic acid, 300 mL of dichloromethane and 100 mL of ultrapure water, and stir vigorously. Remove the water layer, add 100mL ultrapure water, and wash twice in the same way. The resulting precipitate was filtered off, washed with dichloromethane and dried under reduced pressure. The solvent in the filtrate was distilled off, dichloromethane was added to the resulting precipitate, and the precipitate was recovered in the same manner as above. All recovered precipitates were combined, suspended in 80 mL of acetonitrile, and stirred vigorously. To this was added 3.0 mL of tris(2-aminoethyl)amine (molecular weight 146.23, d=0.976) at one time, and stirred at 25° C. for 10 minutes. Then, 4.8 mL of ethyl trifluoroacetate (molecular weight 142.08, d=1.194) was added, and 5.6 mL of triethylamine (molecular weight 101.19, d=0.726) was added, and stirred at 25° C. for 3 hours. The solvent was distilled off and purified by silica gel column (5-10% MeOH/CH ₂ Cl ₂ ). The solvent was distilled off, dissolved in a small amount of acetone, and a white precipitate was formed when diethyl ether was added, filtered, washed with diethyl ether, and dried under reduced pressure to obtain 884 mg (33.5%) of the target substance (compound 102).

In addition, when the synthesis was carried out in the same manner as the above-mentioned method except that the amount of raw materials, solvents, etc. used, reaction time and steps were slightly changed, the yield could be increased to 37%. That is, 597 mg (2.0 mmol) of (E)-5-(2-carboxyvinyl)-2'-deoxyuridine 101 (molecular weight 298.25), 460 mg (4.0 mmol) of N-hydroxysuccinimide (molecular weight 115.09) and 767mg (4.0mmol) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (molecular weight 191.70) were added into the flask with a stirring bar, added 5.0mL of DMF, and stirred at 25°C for 3 Hour. Add about 0.5mL acetic acid, add 100mL dichloromethane and 100mL ultrapure water, and stir vigorously. The resulting precipitate was filtered off, washed with water, and dried under reduced pressure overnight. The obtained white residue was suspended in 50 mL of acetonitrile and stirred vigorously. 3.0 mL (20 mmol) of tris(2-aminoethyl)amine (molecular weight: 146.23, d=0.976) was added at one time, and stirred at 25° C. for 10 minutes. Then, 4.8 mL of ethyl trifluoroacetate (molecular weight 142.08, d=1.194) was added, and 5.6 mL (40 mmol) of triethylamine (molecular weight 101.19, d=0.726) was added, and stirred at 25° C. for 16 hours. The solvent was distilled off and purified by silica gel column (5-10% MeOH/CH ₂ Cl ₂ ). The solvent was distilled off, dissolved in a small amount of acetone, and a white precipitate was formed when ether was added, filtered, washed with ether, and dried under reduced pressure to obtain 453 mg (37%) of the target substance (compound 102) as a white powder. The instrumental analysis values of Compound 102 are shown below, and the ¹ H NMR spectrum is shown in FIG. 2 .

2-[2-[N,N-bis(2-trifluoroacetylaminoethyl)]-aminoethyl]carbamoyl-(E)-vinyl]-2'-deoxyuridine (compound 102):

¹ HNMR (CD ₃ OD): δ8.35(s, 1H), 7.22(d, J=15.6Hz, 1H), 7.04(d, J=15.6Hz, 1H), 6.26(t, J=6.6Hz, 1H), 4.44-4.41(m, 1H), 3.96-3.94(m, 1H), 3.84(dd, J=12.2, 2.9Hz, 1H), 3.76(dd, J=12.2, 3.4Hz, 1H), 3.37 -3.30 (m, 6H), 2.72-2.66 (m, 6H), 2.38-2.23 (m, 2H). ¹³ CNMR (CD ₃ OD): δ169.3, 163.7, 159.1 (q, J=36.4Hz), 151.2, 143.8, 134.3, 122.0, 117.5 (q, J=286Hz), 110.9, 89.1, 87.0, 71.9, 62.5, 54.4, 53.9, 41.7, 38.9, 38.7. C ₂₂ H ₂₉ F ₆ N ₆ O ₈ ([M +H] ⁺ ) has a calculated HRMS (ESI) value of 619.1951 and a measured value of 619.1943.

Example 2: 5'-O-dimethoxytrityl-(2-[2-[N,N-bis(2-trifluoroacetylaminoethyl)]-aminoethyl]carbamoyl- (E)-vinyl)-2'-deoxyuridine (5'-O-DMTr-(2-[2-[N, N-bis(2-trifluoroacetamidoethyl)]-aminoethyl]carbamoyl-(E)-vinyl )-2'-deoxyuridine, the synthesis of compound 103)

The 5'-hydroxyl of compound 102 was protected with dimethoxytrityl (DMTr) group to obtain compound 103. That is, first, 618 mg of compound 102 (molecular weight 618.48) and 373 mg of 4,4'-dimethoxytrityl chloride (molecular weight 338.83) were added to a flask containing a stirring bar, 10 mL of pyridine was added, and the mixture was heated at 25° C. Stir for 16 hours. A small amount of water was added, the solvent was distilled off, and purified with a silica gel column (2-4% MeOH, 1% Et ₃ N/CH ₂ Cl ₂ ). The solvent in the fraction containing the target compound 103 was distilled off to obtain 735.2 mg (79.8%) of the target compound (compound 103). The instrumental analysis values of Compound 103 are shown below, and the ¹ H NMR spectrum is shown in FIG. 3 .

5'-O-Dimethoxytrityl-(2-[2-[N,N-bis(2-trifluoroacetylaminoethyl)]-aminoethyl]carbamoyl-(E)- Vinyl)-2'-deoxyuridine (compound 103):

¹ HNMR (CD ₃ OD): δ7.91(s, 1H), 7.39-7.11(m, 9H), 7.02(d, J=15.6Hz, 1H), 6.93(d, J=15.6Hz, 1H), 6.80-6.78(m, 4H), 6.17(t, J=6.6Hz, 1H), 4.38-4.35(m, 1H), 4.06-4.04(m, 1H), 3.68(s, 6H), 3.32-3.22( m, 8H), 2.66-2.55 (m, 6H), 2.40 (ddd, J=13.7, 5.9, 2.9Hz, 1H), 2.33-2.26 (m, 1H). ¹³ CNMR (CD ₃ OD): δ168.9 , 163.7, 160.1, 159.1 (q, J=36.9Hz), 151.0, 146.1, 143.0, 137.0, 136.9, 134.1, 131.24, 131.16, 129.2, 128.9, 128.0, 122.5, 117.5 (q, J=286.7Hz), 114. , 110.9, 88.1, 87.9, 87.6, 72.6, 65.0, 55.7, 54.2, 53.9, 41.7, 38.9, 38.6. Calcd HRMS (ESI) value of _{C 43} H ₄₇ F ₆ N ₆ O ₁₀ ([M+H] ⁺ ) It is 921.3258, and the measured value is 921.3265.

Example 3: '-O-(dimethoxytrityl)-(2-[2-[N,N-bis(2-trifluoroacetylaminoethyl)]-aminoethyl]carbamoyl -(E)-vinyl)-2'-deoxyuridine, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (5'-O-DMTr -(2-[2-[N,N-bis(2-trifluoroacetamidoethyl)]-aminoethyl]carbamoyl-(E)-vinyl)-2′-deoxyuridine, 3′-[(2-cyanoethyl)-(N,N -diisopropyl)]-phosphoramidite, compound 104) synthesis

188mg (0.20mmol) of compound 103 (molecular weight 920.85) was azeotroped with CH ₃ CN, 28.6mg (0.40mmol) of 1H-tetrazole (molecular weight 70.05) was added, and dried overnight with a vacuum pump. Add 5.1mL CH ₃ CN, stir after dissolving the reagent, add 194μL (0.60mmol) 2-cyanoethyl-N,N,N',N'-tetraisopropylphosphoramidite (molecular weight 301.41, d=0.949 ), stirred at 25°C for 2 hours. A mixture of 50 mL of ethyl acetate and 50 mL of saturated aqueous sodium bicarbonate solution was added to separate the layers, and the organic layer was washed with saturated brine and dried over magnesium sulfate. After removing magnesium sulfate by filtration, the solvent was distilled off. After azeotroping the crude product obtained by liquid separation with CH ₃ CN, a 0.1M CH ₃ CN solution was prepared assuming that the yield of the obtained product (compound 104) was 100%, and used for DNA synthesis. In addition, it was confirmed that compound 104 was obtained according to ³¹ PNMR (CDCl ₃ ) and HRMS (ESI) of the crude product. Its value is shown below.

Compound 104:

³¹ PNMR (CDCl ₃ ) δ149.686, 149.430; HRMS (ESI) calculated for C ₅₂ H ₆₄ F ₆ N ₈ O ₁₁ P ([M+H] ⁺ ) 1121.4336, found 1121.4342.

Example 4: Synthesis of DNA Oligomers

[Chemical 54]

Scenario 2

The synthesis of oligomeric DNA using compound 104 by means of an automatic DNA synthesizer was carried out by the conventional phosphoramidite method (DMTr OFF) on a 1 μmol scale, and a compound called 5'-d(CGCAATXTAACGC)-3'(13-mer, X The DNA oligomer of the sequence (SEQ ID NO.1) whose structure is shown in chemical formula 105). Deprotection was carried out at 55° C. for 16 hours with concentrated aqueous ammonia (28% by mass). Use Speed Vac to volatilize ammonia, pass through a 0.45 μm filter, analyze the DNA oligomers retained by reverse phase chromatography, and purify the peak that appears at about 10.5 minutes (CHEMCOBOND 5-ODS-H (trade name); 10×150 mm, 3 mL/min, 5-30% CH ₃ CN/50 mM TEAA buffer pH 7 (20 min), detection at 260 nm). The molecular weight of the purified product was determined by the negative mode of MALDI TOF mass spectrometry, and it was confirmed that it had the expected molecular weight (based on C ₁₃₄ H ₁₇₆ N ₅₂ O ₇₆ P ₁₂ , calcd. 4102.8) ([MH] ^- found 4101.9, calcd. 4101.8). The MALDI TOF mass spectrum is shown in Figure 4.

In addition, the 5'-d(CGCAATXTAACGC)-3' sequence (13-mer, the structure of X is shown in chemical formula 105) can be synthesized in the same way as above, except that it is deprotected with concentrated ammonia or heated at 55°C After 4 hours, it was carried out at 25°C for 16 hours. The concentration of TEAA (triethylamine acetate) buffer (pH 7) in reversed-phase HPLC was 0.1M, and the development time was 30 minutes or more in reversed-phase HPLC. . And, by the same method, the DNA (containing the nucleotide represented by chemical formula 105) shown in Table 1 as the raw material of each ODN can be synthesized.

In order to measure the concentration of each synthesized DNA, each purified DNA was treated with calf intestinal alkaline phosphatase (50 U/mL), snake venom phosphodiesterase (0.15 U/mL) and P1 nuclease (50 U/mL) at 25°C 16 hours for complete digestion. The resulting digested solution was analyzed by HPLC using a CHEMCOBOND 5-ODS-H (trade name) column (4.6×150 mm). At this time, 0.1 MTEAA (pH 7.0) was used as a developing solution at a flow rate of 1.0 mL/min. The concentration of the synthesized DNA was determined by comparison with the peak areas of standard solutions containing dA, dC, dG and dT each at a concentration of 0.1 mM. And, the synthetic DNA was also determined by MALDI TOF mass spectrometry. The mass analysis values are shown below. In addition, [105] indicates that the nucleotide represented by Chemical Formula 105 is inserted at this position.

CGCAAT[105]TAACGC, calculated for C ₁₃₄ H ₁₇₇ N ₅₂ O ₇₆ P ₁₂ ([M+H] ⁺ ) 4103.8, found 4107.0;

TTTTTT[105]TTTTTT, calculated for C ₁₃₈ H ₁₈₇ N ₃₀ O ₉₀ P ₁₂ ([M+H] ⁺ ) 4077.8, found 4076.9;

TGAAGGGCTT[105]TGAACTCTG, calculated for C ₂₀₅ H ₂₆₅ N ₇₇ O ₁₂₂ P ₁₉ ([M+H] ⁺ ) 6348.2, found 6348.7;

GCCTCCT[105]CAGCAAATCC[105]ACCGGCGTG, calculated for C ₂₈₅ H ₃₇₆ N ₁₀₈ O ₁₆₉ P ₂₇ ([M+H] ⁺ ) 8855.0, found 8854.8;

CCTCCCAAG[105]GCTGGGAT[105]AAAGGCGTG, calculated for C ₂₈₉ H ₃₇₆ N ₁₁₆ O ₁₆₈ P ₂₇ ([M+H] ⁺ ) 8999.1, found 9002.2.

Example 5: Biotin modification of DNA oligomers containing nucleotides with 2 amino groups

[Chemical 55]

Option 3

By reacting the synthetic DNA oligomer 5'-d(CGCAATXTAACGC)-3' (compound 105, using the compound 4 as X) with N-hydroxysuccinimide ester of biotin, the 2 amino groups were converted to 2 biotin labels (Scheme 3 above). That is, first, mix 30 μL of 5'-d(CGCAATXTAACGC)-3' (compound 105, chain concentration 320 μM), 10 μL of Na ₂ CO ₃ /NaHCO ₃ buffer (1M, pH 9.0) and 60 μL of H ₂ O, and add 100 μL of biological DMF solution (20 mM) of the N-hydroxysuccinimide ester of the element, and mix well. After standing at 25°C for 16 hours, add 800 μL H ₂ O, pass through a 0.45 μm filter, and purify the peak that appears at about 14 minutes by reverse-phase HPLC (CHEMCOBOND 5-ODS-H; 10×150 mm, 3 mL/ min, 5-30% CH ₃ CN/50 mM TEAA buffer (20 min, detection at 260 nm). As a result of measuring the product obtained by this HPLC purification by MALDI TOF mass spectrometry, a peak was observed at 4554.3. This peak is calculated from the [MH] calculated by the molecular weight of 4555.4 (calculated value obtained from C ₁₅₄ H ₂₀₄ N ₅₆ O ₈₀ P ₁₂ S ₂ ) of the target product 6 obtained by reacting two amino groups with two biotin molecules ^. The value 4554.4 matches. The MALDI TOF mass spectrum is shown in Figure 5.

Double-stranded DNA and RNA were synthesized using this compound 6 (single-stranded state), and the fluorescence intensities of the single-stranded state and the double-stranded state were compared. As a result, it was confirmed that the fluorescence emission of the DNA fluorescent probe (compound 6) was suppressed in a single-stranded state, and strong fluorescence was emitted when forming a double helix with a complementary nucleic acid.

In the following examples 6-13, thiazole orange derivatives with carboxymethylene linkers shown in the following chemical formulas b and c were synthesized and activated into N-hydroxysuccinates by combining them with active amino groups Various fluorescent oligonucleotides (fluorescent DNA probes) were prepared by reacting DNA oligomers (oligonucleotides). That is, various oligonucleotides (fluorescent DNA probes) having various changes in the length of the methylene linker extending from the dye and the linker containing an amino group extending from the 5-position of thymidine were produced . As a result, in any of the various fluorescent DNA probes, the fluorescence emission of the DNA fluorescent probe in a single-stranded state can be suppressed, and can emit strong fluorescence when forming a double helix with a complementary nucleic acid. In addition, in the following chemical formulas b and c, n represents the length of the linker (number of bridging atoms).

[Chemical 56]

Example 6: Synthesis of Compounds Having Thiazole Orange-Derived Structures at Two Positions in One Molecule

[Chemical 57]

Option 4

As in Scheme 4 above, a DNA oligomer (oligonucleotide) 110 having a thiazole orange-derived structure at two positions in one molecule was synthesized. More details are described below.

The synthesis of thiazole orange derivative 107 refers to Organic Letters 2000, 6, 517-519, as described in Scheme 5 below.

[Chemical 58]

Option 5

(1) Synthesis of N-methylquinolinium iodide (compound 111)

First, N-methylquinolinium iodide (compound 111) was synthesized according to the description in the above document. Specifically, 2.4 mL of quinoline and 4 mL of methyl iodide were added to 42 mL of anhydrous dioxane, and after stirring at 150 ° C for 1 hour, the precipitate was collected by filtration, washed with diethyl ether and petroleum ether, and dried to obtain N-iodide Methylquinolinium (Compound 111).

(2) Synthesis of 3-(4-carboxybutyl)-2-methylbenzothiazolium bromide (compound 112)

8 mL of 2-methylbenzothiazole (FW149.21, d=1.173) and 9.4 g of 5-bromovaleric acid (FW181.03) were stirred at 110° C. for 16 hours. The crude product was cooled to room temperature, the resulting solid was suspended in 20 mL of methanol, and 40 mL of ether was added. The resulting precipitate was filtered out, washed with dioxane until the smell of 2-methylbenzothiazole disappeared, washed with diethyl ether, and dried under reduced pressure to obtain 9.8 g of white powder. ¹ H NMR of the white powder was measured, and it was found that 3-(4-carboxybutyl)-2-methylbenzothiazolium bromide (compound 112), the target product of which the 2-position was alkylated, and the 2-position was not alkylated A mixture of 3-(4-carboxybutyl)-benzothiazolium bromide. The ratio of proton peaks was: unalkylated species: alkylated species = 10:3. The crude product was used directly in subsequent reactions.

(3) Synthesis of brominated 1-methyl-4-[{3-(4-carboxybutyl)-2(3H)-benzothiazolylidene}methyl]quinoline (compound 107)

In the presence of 3.6 ml of triethylamine (FW101.19, d=0.726), the 3-(4-carboxybutyl)-2-methylbenzothiazolium bromide ( 2.18 g of the crude product of compound 112) and 700 mg of iodized N-methylquinoline (compound 111) (FW271.10) were stirred in 10 mL of dichloromethane at 25° C. for 2 hours. Subsequently, 50 ml of diethyl ether was added, and the resulting precipitate was filtered, washed with diethyl ether, and dried under reduced pressure. The precipitate was suspended in 50 mL of ultrapure water, filtered, washed with ultrapure water, and dried under reduced pressure. The above precipitate was suspended in 50 mL of acetonitrile, filtered, washed with acetonitrile, and dried under reduced pressure to obtain 307.5 mg of a red powder (25.3% yield). Comparing the ¹ HNMR spectrum with the reference value, it was determined that the red powder was the target product (compound 107).

In addition, 3-(4-carboxybutyl)-2-methylbenzothiazolium bromide (compound 112) and 1-methyl-4-[{3-(4-carboxybutyl)-2( 3H)-Benzothiazolylene}methyl]quinoline (compound 107) can also be synthesized in the following manner. That is, first, 11.7 mL (92 mmol) of 2-methylbenzothiazole (FW149.21, d=1.173) and 13.7 g (76 mmol) of 5-bromovaleric acid (FW181.03) were stirred at 150°C for 1 Hours. The crude product was cooled at room temperature, the resulting solid was suspended in 50 ml of methanol, and 200 ml of ether was added. The resulting precipitate was filtered, washed with ether, and dried under reduced pressure to obtain 19.2 g of lavender powder. This powder was a mixture of target compound 112 (3-(4-carboxybutyl)-2-methylbenzothiazolium bromide) and 2-methylbenzothiazolium bromide. ¹ H NMR (in DMSO-d6) measured the mixture, from the peak area of the peak at 8.5ppm (from the target compound 112) and the peak at 8.0ppm (from 2-methylbenzothiazolium bromide) The yield of the target compound 112 was calculated to be 9.82 g (14 mmol, 32%). This mixture (crude product) was used in subsequent reactions without purification. In addition, 5-bromopentanoic acid was replaced by 4-bromobutyric acid, and the brominated 3-(4- Carboxypropyl)-2-methylbenzothiazolium obtained in 4% yield. In addition, 5-bromopentanoic acid was replaced by 6-bromohexanoic acid, and the brominated 3-(4- Carboxypentyl)-2-methylbenzothiazolium obtained in 35% yield. Furthermore, 5-bromopentanoic acid was replaced by 7-bromoheptanoic acid, and the brominated 3-(4 -carboxyhexyl)-2-methylbenzothiazolium obtained in 22% yield.

Subsequently, to 3.24 g of the above mixture (crude product) containing compound 112 (3-(4-carboxybutyl)-2-methylbenzothiazolium bromide) and 2-methylbenzothiazolium bromide was added 1.36 g (5.0 mmol) of iodide N-methylquinoline (compound 111) (FW271.10), 7.0 mL (50 mmol) of triethylamine (FW101.19, d=0.726) and 100 mL of dichloromethane, A clear solution was thus obtained. The solution was stirred at 25°C for 16 hours. Subsequently, the solvent was distilled off under reduced pressure. Acetone (200 mL) was added to the residue, and the resulting precipitate was filtered, followed by washing with acetone. The residue thus obtained was dried under reduced pressure, and the dried red residue was washed with distilled water (50 mL). The residue was filtered again, washed with distilled water, and dried under reduced pressure to obtain the target product (compound 107) (654 mg, 1.39 mmol, 28%) as a red powder. Comparing the ¹ H NMR spectrum with the reference value confirmed that the red powder was the target product (compound 107). Peaks of ¹ H NMR and ¹³ C NMR (DMSO-d6) and measured values of HRMS (ESI) are shown below. In addition, FIG. 6 shows the ¹ HNMR spectrum (DMSO-d6) of Compound 107.

Compound 107: ¹ HNMR (DMSO-d6): δ8.74 (d, J=8.3Hz, 1H), 8.51 (d, J=7.3Hz, 1H), 7.94-7.89 (m, 3H), 7.74-7.70( m, 1H), 7.65(d, J=8.3Hz, 1H), 7.55-7.51(m, 1H), 7.36-7.32(m, 1H), 7.21(d, J=7.3Hz, 1H), 6.83(s , 1H), 4.47(t, J=7.1Hz, 2H), 4.07(s, 3H), 2.22(t, J=6.6Hz, 1H), 1.77-1.63(m, 4H); ¹³ CNMR (DMSO-d6 , 60°C) δ174.6, 158.8, 148.4, 144.5, 139.5, 137.6, 132.7, 127.9, 126.8, 125.5, 124.1, 123.7, 123.6, 122.4, 117.5, 112.6, 107.6, 87.4, 45.6, 45.0, 32.5 22.3; HRMS ( _{ESI )} calcd for _C23H23N2O2S ([M.Br] ⁺ ₎ 391.1480, found 391.1475.

In addition, using the same method as for the preparation of the above-mentioned compound 107, a compound having Brominated 4-((3-(3-carboxypropyl)benzo[d]thiazole-2(3H)-ylidene) of a linker (polymethylene chain connected to carboxyl group) with carbon number n=3 ) methyl)-1-methylquinoline with a yield of 43%. Instrumental analysis values are shown below.

4-((3-(3-carboxypropyl)benzo[d]thiazol-2(3H)-ylidene)methyl)-1-methylquinoline bromide:

¹ H NMR (DMSO-d6) δ8.85 (d, J = 8.3Hz, 1H), 8.59 (d, J = 7.3Hz, 1H), 8.02.7.93 (m, 3H), 7.78.7.70 (m, 2H) , 7.61.7.57(m, 1H), 7.42.7.38(m, 1H), 7.31(d, J=6.8Hz, 1H), 7.04(s, 1H), 4.47(t, J=8.1Hz, 2H), 4.13 (s, 3H), 2.52.2.48 (m, 2H), 1.99.1.92 (m, 2H); ¹³ CNMR (DMSO-d6, 60°C) δ174.3, 158.9, 148.6, 144.5, 139.5, 137.7, 132.7 , 127.9, 126.7, 125.6, 124.1, 124.0, 123.7, 122.5, 117.5, 112.5, 107.6, 87.7, 45.6, 42.0, 31.6, 22.4; HRMS of C ₂₂ H ₂₁ N ₂ O ₂ S ([M.Br] ⁺ ) (ESI) calculated value is 377.1324, found value is 377.1316.

In addition, using the same method as for the preparation of the above-mentioned compound 107, a compound having Brominated 4-((3-(3-carboxypentyl)benzo[d]thiazole-2(3H)-ylidene of a linker (polymethylene chain connected to carboxyl group) with carbon number n=5 ) methyl)-1-methylquinoline with a yield of 26%. Instrumental analysis values are shown below.

4-((3-(3-carboxypentyl)benzo[d]thiazol-2(3H)-ylidene)methyl)-1-methylquinoline bromide:

¹ H NMR (DMSO-d6) δ8.70 (d, J = 8.3Hz, 1H), 8.61 (d, J = 6.8Hz, 1H), 8.05.8.00 (m, 3H), 7.80.7.73 (m, 2H) , 7.60.7.56(m, 1H), 7.41.7.35(m, 2H), 6.89(s, 1H), 4.59(t, J=7.3Hz, 2H), 4.16(s, 3H), 2.19(t, J =7.3Hz, 1H), 1.82.1.75(m, 2H), 1.62.1.43(m, 4H); ¹³ CNMR (DMSO-d6, 60°C) δ174.5, 159.0, 148.6, 144.7, 139.7, 137.8, 132.9 , 127.9, 126.9, 125.2, 124.2, 123.8, 123.6, 122.6, 117.8, 112.6, 107.7, 87.4, 45.6, 42.1, 36.0, 26.3, 25.9, 24.9; C ₂₄ H ₂₅ N ₂ O ₂ S ([M.Br] +) has a calculated HRMS (ESI) value of 405.1637 and a measured value of 405.1632.

In addition, using the same method as for the preparation of the above-mentioned compound 107, a compound with carbon Brominated 4-((3-(3-carboxyhexyl)benzo[d]thiazole-2(3H)-ylidene)methylidene of a linker (polymethylene chain connected to carboxyl group) with atomic number n=6 base)-1-methylquinoline in 22% yield. Instrumental analysis values are shown below.

4-((3-(3-carboxyhexyl)benzo[d]thiazol-2(3H)-ylidene)methyl)-1-methylquinoline bromide:

¹ HNMR (DMSO-d6) δ8.72(d, J=8.3Hz, 1H), 8.62(d, J=6.8Hz, 1H), 8.07.8.01(m, 3H), 7.81.7.75(m, 2H) , 7.62.7.58(m, 1H), 7.42.7.38(m, 2H), 6.92(s, 1H), 4.61(t, J=7.3Hz, 2H), 4.17(s, 3H), 2.18(t, J =7.3Hz, 1H), 1.82.1.75(m, 2H), 1.51.1.32(m, 6H); ¹³ CNMR (DMSO-d6, 60°C) δ174.0, 159.1, 148.6, 144.7, 139.8, 137.8, 132.9 , 127.9, 126.8, 125.0, 124.2, 123.8, 123.6, 122.6, 118.0, 112.7, 107.8, 87.4, 45.5, 42.1, 33.4, 27.9, 26.4, 25.5, 24.1; C ₂₅ H ₂₇ N ₂ O ₂ S ([M. Br]+) has a calculated HRMS (ESI) value of 419.1793 and an observed value of 419.1788.

(4) Synthesis of N-hydroxysuccinimide ester 109

9.4 mg (20 μmol) of brominated 1-methyl-4-[{3-(4-carboxybutyl)-2(3H)-benzothiazolyl}methyl]quinoline (compound 107) (FW471 .41), 4.6 mg (40 μmol) of N-hydroxysuccinimide (compound 108) (FW115.09) and 7.6 mg (40 μmol) of EDC (1-ethyl-3-(3-dimethylaminopropyl base) carbodiimide hydrochloride) (FW191.70) was stirred in 1 mL of DMF at 25°C for 16 hours to obtain the N-hydroxysuccinimide ester (compound 109) in which the carboxyl group of the dye (compound 107) was activated ). This reaction product was not purified, and the reaction solution (dye 20 mM) was directly used for the reaction with oligo DNA (oligonucleotide).

In addition, except that a compound having a linker (polymethylene chain) with a different number of carbon atoms was used instead of compound 107 as a raw material, the same method as that used to prepare compound 109 was used to synthesize a linker with n=3 carbon atoms ( brominated 4-((3-(4-(succinimidyloxy)-4-oxobutyl)benzo[d]thiazole-2(3H)-ylidene)methylidene) base)-1-methylquinoline. In addition, brominated 4-((3-(4-(succinimidyloxy)-4-oxygen) having a linker (polymethylene chain) with carbon number n=5 was also synthesized in the same manner. Bromination of hexyl)benzo[d]thiazole-2(3H)-ylidene)methyl)-1-methylquinoline and a linker (polymethylene chain) with carbon number n=6 -((3-(4-(Succinimidyloxy)-4-oxoheptyl)benzo[d]thiazol-2(3H)-ylidene)methyl)-1-methylquinoline.

(5) Synthesis of DNA oligomer (oligonucleotide 110) modified with 2 thiazole orange molecules

A DNA oligomer (oligonucleotide) 105 having two active amino groups was synthesized by a conventional method using an automatic DNA synthesizer as in Example 4 above. The sequence of compound 105 is 5'-d(CGCAATXTAACGC)-3' the same as that of Example 4 (X is the aforementioned compound 104). Subsequently, this DNA oligomer (oligonucleotide) 105 was reacted with N-hydroxysuccinimide ester (compound 109), and a DNA oligomer having a structure derived from thiazole orange at two positions in one molecule was synthesized. Objects (oligonucleotides)110. That is, first, 30 μL of 5′-d(CGCAATXTAACGC)-3′ (compound 105, chain concentration 320 μM), 10 μL of Na ₂ CO ₃ /NaHCO ₃ buffer (1M, pH 9.0) and 60 μL of H ₂ O to mix, then add 100 μL of N-hydroxysuccinimide ester (compound 109) in DMF (20 mM) and mix well. Stand at 25°C for 16 hours, then add 800 μL H ₂ O, pass through a 0.45 μm filter, and purify the peak that appears in reverse chromatography at about 14.5 minutes (CHEMCOBOND 5-ODS-H 10×150mm, 3mL/min, 5- 30% _CH3CN /50 mM TEAA buffer (20 min), detection at 260 nm). Figure 7 shows the reverse HPLC profile. Fractions indicated by peaks indicated by arrows were separated and purified. The HPLC-purified product was measured by the negative mode of MALDI TOF mass spectrometry, and a peak at 4848.8 was found, which was identified as DNA oligomer (oligonucleotide) 110 . FIG. 8 shows the MALDI TOF MASS spectrum of DNA oligomer (oligonucleotide) 110 . In this figure, the arrow indicates the mass peak (4848.8) of the product obtained in the above-mentioned purification. This peak is consistent with ^the calculated value of 4848.8 for [M ²⁺ -3H ^{+ ] -} composed of molecules of DNA oligomers (oligonucleotides) ¹¹⁰ with 2 positive charges M(C ₁₈₀ H ₂₂₀ N ₅₆ O ₇₈ P ₁₂ S ₂ ) was obtained by removal of 3 protons. In addition, the peaks on the left and right sides are peaks derived from DNA T8-mer and T18-mer added as a standard.

Example 7: Use of DNA oligomers (oligonucleotides) 110 as fluorescent probes

The DNA oligomer (oligonucleotide) 110 (DNA with 2 dye molecules) purified in Example 6 was desalted, freeze-dried, and then prepared into an aqueous solution, and its concentration was determined by UV absorption (X is approximate to T). Thereafter, under the conditions of a strand concentration of 2.5 μM, a phosphate buffer of 50 mM (pH 7.0), and NaCl of 100 mM, when the fluorescent probe (DNA oligomer 110) is in a single-stranded state, it becomes a DNA-DNA double helix UV measurement was performed separately when it was a DNA-RNA double helix. Figure 9 shows the spectra of these three samples. In this figure, the dotted line represents the spectrum of the fluorescent probe in a single-stranded state, the thick line represents the spectrum of the DNA-DNA duplex, and the thin line represents the spectrum of the DNA-RNA duplex. As can be seen from the figure, the maximum wavelength of UV absorption around 500 nm shifts due to the formation of a double helix. In addition, in FIG. 9 and all other UV absorption spectra, the horizontal axis represents wavelength (nm) and the vertical axis represents absorbance.

Subsequently, under the same conditions of a chain concentration of 2.5 μM, a phosphate buffer of 50 mM (pH 7.0) and a NaCl of 100 mM, excitation by excitation light of 488 nm (bandwidth 1.5 nm) was performed, followed by fluorescence measurement. Fig. 10 shows the spectra of three samples in which the fluorescent probe is single-stranded (dotted line), DNA-DNA double helix (thick line) and DNA-RNA double helix (thin line), respectively. It can be seen from the figure that, compared with the fluorescence intensity of the single-stranded fluorescent probe at 530 nm, the fluorescence intensity of DNA-DNA increases by 15 times, and that of DNA-RNA increases by 22 times. In addition, in FIG. 10 and all other fluorescence emission spectra and excitation spectra, the horizontal axis represents the wavelength (nm), and the vertical axis represents the emission intensity.

In addition, the same result was also obtained by using excitation light of 510 nm instead of excitation light of 488 nm. Figure 11 shows its spectrum.

Example 8: Synthesis of Compounds with Linkers of Various Lengths and Use as Fluorescent Probes

Compounds (DNA oligomers) represented by the following Chemical Formula 113 in which the linker length n was varied in various ways were synthesized. The synthesis is the same as in the above-mentioned Examples 1-4 and 6, except that the raw material 5-bromovaleric acid is replaced with a compound having a changed number of carbon atoms (chain length) in order to satisfy the length of the linker. In this example, the sequence of the following compound 113 is 5'-d(CGCAATXTAACGC)-3' (X is the dye-introducing moiety). In addition, as in Example 7, each of them was used as a fluorescent probe, and the performance was evaluated by fluorescence measurement. As a result, it was confirmed that the fluorescence increased by about 10 times or more by hybridizing with the target nucleic acid as compared with the single-stranded probe within the length range of the linker shown below. In addition, the duplex obtained by hybridizing the probe to the target nucleic acid has higher thermal stability than the duplex of the natural sequence.

Chemical 59

Table 1

5′-d(CGCAATTTAACGC)-3′/5′-d(GCGTTAAATTGCG)-3′Tm(℃) 58

5′-d(CGCAATTTAACGC)-3′/5′-r(GCGUUAAAUUGCG)-3′Tm(℃) 46

Determination conditions: probe (compound 113) 2.5 μM, phosphate buffer 50 mM (pH7.0), NaCl 100 mM, complementary chain 2.5 μM

The maximum wavelength of fluorescence is a value when excited with light of 488 nm (bandwidth: 1.5 nm).

Quantum yields were calculated using 9,10-diphenylanthracene as a reference.

FIG. 12 shows the absorption spectrum when the linker length n=4 in Example 8. The dotted line is the spectrum of single strand, the solid line is the spectrum of DNA-DNA strand, and the dotted line is the spectrum of DNA-RNA strand. When the absorption at 400-600 nm was observed, the absorption band in the single-chain state appeared on the short wavelength side compared with the absorption band after hybridization, clearly indicating that H-aggregates were formed by the dye dimer in the single-chain state.

In addition, FIG. 13 also shows the excitation spectrum and fluorescence emission spectrum when the linker length n=4 in Example 8. In this figure, the curve on the left (short wavelength side) represents the excitation spectrum, and the curve on the right (long wavelength side) represents the fluorescence emission spectrum. In addition, in this figure, the parentheses of the legend indicate the reference fluorescence emission wavelength (fluorescence λ _max ) in the excitation spectrum and the excitation wavelength in the fluorescence emission spectrum, respectively. In the excitation spectrum and the fluorescence emission spectrum, the luminescence intensity of the single strand is the weakest, that of the DNA-DNA strand is slightly stronger, and that of the DNA-RNA strand is the strongest. It can be seen from the excitation spectrum that the absorption related to the fluorescence emission is only the absorption band on the long wavelength side in FIG. 12 , while the absorption on the short wavelength side is not related to the fluorescence emission. In other words, it was clearly shown that fluorescence emission can be controlled through the exciton effect. Therefore, the fluorescence emission is enhanced after hybridization and is extremely weak in the single-stranded state. Therefore, the states before and after hybridization can be clearly distinguished.

Example 9

A compound (DNA oligomer) having only one dye structure in one molecule represented by the following chemical formula 114 in which the linker length n varied in various ways was synthesized. The synthesis is the same as the above-mentioned Examples 1-4 and 6, except that in order to meet the length of the linker, the raw material 5-bromovaleric acid is replaced by a compound with a changed number of carbon atoms (chain length), and in the synthesis of compound 102 , using bis(2-aminoethyl)methylamine instead of tris(2-aminoethyl)amine. Compounds with n=3, 4, 5, and 6 can be synthesized in the same manner.

Chemical 60

More specifically, it was synthesized according to the following scheme. Although the following schemes are for the case of n=4, they can also be synthesized similarly when n is other numerical values.

Chemical 61

(E)-5-(3-(2-(N-methyl-N-(2-(2,2,2-trifluoroacetylamino)ethyl)amino)ethylamino)-3-oxopropane -1-enyl)-2′-deoxyuridine ((E)-5-(3-(2-(N-Methyl-N-(2-(2,2,2-trifluoroacetamido)ethyl)amino)ethylamino )-3-oxoprop-1-enyl)-2′-deoxyuridine)(102′) Synthesis

1.19g (4.0mmol) of (E)-5-(2-carboxyvinyl)-2'-deoxyuridine 101 (molecular weight 298.25) and 921mg (8.0mmol) of N-hydroxysuccinimide (molecular weight 115.09 ) and 1.53g (8.0mmol) of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (molecular weight 191.70) were added to the recovery flask that had been placed in the stirring bar, and then 1.0mL of DMF, stirred at 25°C for 8 hours. About 1 mL of acetic acid was added, and 250 mL of dichloromethane and 250 mL of ultrapure water were added, followed by vigorous stirring. The resulting precipitate was filtered, washed with water, and dried overnight under reduced pressure. The resulting white residue was suspended in 100 mL of acetonitrile and stirred vigorously. 2.34 g (20 mmol) of N-methyl-2,2'-diaminodiethylamine (molecular weight 146.23, d=0.976) was added thereto at one time, and stirred at 25° C. for another 10 minutes. Thereafter, 4.8 mL (40 mmol) of ethyl trifluoroacetate (molecular weight 142.08, d=1.194), 5.6 mL (40 mmol) of triethylamine (molecular weight 101.19, d=0.726) and 50 mL of ethanol were added and stirred at 25°C 16 hours. The solvent was distilled off from the resulting mixture under reduced pressure, and purified with a silica gel column (10-20% MeOH/CH ₂ Cl ₂ ). The solvent was distilled off under reduced pressure from the fraction containing the desired product, and the mixture was dissolved in a small amount of acetone. When diethyl ether was added, a white precipitate formed. After filtration, washing with ether, and drying under reduced pressure, 750 mg (76%) of the desired product (compound 102') was obtained as a white powder. Instrumental analysis values are shown below. Compound 102':

¹ HNMR (CD ₃ OD) δ8.29(s, 1H), 7.17(d, J=15.6Hz, 1H), 6.97(d, J=15.6Hz, 1H), 6.21(t, J=6.3Hz, 1H ), 4.40.4.36(m, 1H), 3.92.3.90(m, 1H), 3.80(dd, J=11.7, 2.9Hz, 1H), 3.72(dd, J=11.7, 3.4Hz, 1H), 3.37. 3.25 (m, 5H), 2.60.2.53 (m, 5H), 2.33.2.19 (m, 5H); ¹³ CNMR (CD ₃ OD) δ169.2, 158.7 (q, J=36.4Hz), 151.2, 143.7, 143.6, 134.1, 122.2, 117.5 (q, J=286.2Hz), 111.0, 89.2, 87.0, 72.1, 62.6, 57.4, 56.7, 42.4, 41.8, 38.5, 38.3; C ₁₉ H ₂₇ F ₃ N ₅ O ₇ ([ M+H] ⁺ ) calculated HRMS (ESI) 494.1863, found 494.1854.

(E)-5-(3-(2-(N-methyl-N-(2-(2,2,2-trifluoroacetylamino)ethyl)amino)ethylamino)-3-oxopropane -1-enyl)-5'O-(4,4'-dimethoxytriphenyl)-2'-deoxyuridine 3'O-(2-cyanoethyl)-N,N-diiso Propyl phosphoramidite ((E)-5-(3-(2-(N-Methyl-N-(2-(2,2,2-trifluoroacetamido)ethyl)amino)ethylamino)-3-oxoprop-1- enyl)-5'O-(4,4'-dimethoxytrityl)-2'-deoxyuridine3'O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite) (compound 104') synthesis

First, 296mg (0.60mmol) of compound 102' (molecular weight: 494.19) and 224mg (0.66mmol) of 4,4'-dimethoxytrityl chloride (molecular weight: 338.83) were added to the recovery chamber placed in the stirring bar. In the flask, 4 mL of pyridine was added, and stirred at 25° C. for 2 hours. 1 ml of water was added, the solvent was distilled off under reduced pressure, and purified with a silica gel column (1.5% MeOH and 1% Et ₃ N/CH ₂ Cl ₂ ). The fraction containing the triphenylide of the objective compound 102' (intermediate of compound 104') was concentrated, and saturated aqueous sodium bicarbonate solution was added to the residue. The mixture was extracted with ethyl acetate, washed with saturated brine, and dried under pressure to obtain triphenylide (366 mg, 77%) as white foam.

¹ HNMR (CD ₃ OD) δ7.94(s, 1H), 7.42.7.17(m, 9H), 7.01(d, J=15.6Hz, 1H), 6.95(d, J=15.6Hz, 1H), 6.86 .6.83(m, 4H), 6.21(t, J=6.3Hz, 1H), 4.41.4.38(m, 1H), 4.09.4.06(m, 1H), 3.75(s, 6H), 3.40.3.30(m , 6H), 2.59(t, J=6.8Hz, 2H), 2.53(t, J=6.8Hz, 2H), 2.46.2.31(m, 5H); ¹³ CNMR(CD ₃ OD)δ169.2, 158.7( q, J=36.4Hz), 151.2, 143.7, 143.6, 134.1, 122.2, 117.5 (q, J=286.2Hz), 111.0, 89.2, 87.0, 72.1, 62.6, 57.4, 56.7, 42.4, 41.8, 38.5, 38.3; HRMS (ESI) calcd for C ₄₀ H ₄₅ F ₃ N ₅ O ₉ ([M+H] ⁺ ) 796.3169, found 796.3166.

159 mg (0.20 mmol) of the triphenyl compound (molecular weight: 920.85) of the aforementioned compound 102' and 28.6 mg (0.40 mmol) 1H-tetrazole (molecular weight: 70.05) were put into a round bottom flask, and vacuum-dried overnight with a vacuum pump. At this time, 4.0 mL of CH ₃ CN dissolution reagent was added, followed by stirring, and 191 μL (0.60 mmol) of 2-cyanoethyl-N, N, N', N'-tetraisopropylphosphoramidite (molecular weight 301.41, d=0.949), and stirred at 25°C for 2 hours. After the completion of the reaction was confirmed by TLC, saturated aqueous sodium bicarbonate was added, extracted with ethyl acetate, and the organic layer was washed with saturated brine, followed by drying over magnesium sulfate. After removing magnesium sulfate by filtration, the solvent was distilled off under reduced pressure to obtain a crude product containing the target compound 104'. The composition can be directly used for DNA synthesis without purification. In addition, the acquisition of compound 104' can be confirmed by ³¹ PNMR (CDCl ₃ ) and HRMS (ESI) of the aforementioned crude product. These values are shown below.

Compound 104':

³¹ PNMR (CDCl ₃ ) δ 149.686, 149.393; HRMS (ESI) calculated for C ₄₉ H ₆₁ F ₃ N ₇ O ₁₀ P ([M+H] ⁺ ) 996.4248, found 996.4243.

Synthesis of DNA105' was carried out as in the aforementioned compound 105. Instrumental analysis values are shown below. DNA105': CGCAAT[105']TAACGC, the calculated value of C ₁₃₃ H ₁₇₄ N ₅₁ O ₇₆ P ₁₂ ([M+H] ⁺ ) is 4074.8, the found value is 4072.0; CGCAAT[105'][105']AACGC, Calculated for C ₁₄₀ H ₁₈₇ N ₅₄ O ₇₇ P ₁₂ ([M+H] ⁺ ) 4230.0, found 4228.9.

The synthesis of thiazole orange-introduced DNA 114 was performed as in the aforementioned compound 113. Instrumental analysis values are shown below. CGCAAT[114] ₍₄₎ TAACGC, the calculated value of C ₁₅₆ H ₁₉₄ N ₅₃ O ₇₇ P ₁₂ S(M ⁺ ) is 4447.3, and the measured value is 4445.6; CGCAAT[114] ₍₄₎ [114] ₍₄₎ AACGC, _Calcd for _{C186H228N58O79P12S2} ([MH ^]+ _{) 4976.0} , _{found 4976.9} .

In the synthetic DNA oligomer (ODN), for the ODN (probe containing only one dye) with 5'-d(CGCAAT[114] _(n) TAACGC)-3' sequence, the same as in Examples 7 and 8 Fluorescence changes were observed in that way. Table 2 below, and Figures 14 and 15 show the results. Figure 14 shows the absorption spectrum (the dotted line is the spectrum of single strand, the solid line is the spectrum of DNA-DNA chain, and the dotted line is the spectrum of DNA-RNA chain), and Figure 15 shows the excitation spectrum and emission spectrum. In FIG. 15 , the curve on the left (short wavelength side) represents the excitation spectrum, and the curve on the right (long wavelength side) represents the fluorescence emission spectrum. In addition, in this figure, the wavelengths in the legend represent the wavelength of reference fluorescence emission in the excitation spectrum (λ _max of fluorescence) and the excitation wavelength in the fluorescence emission spectrum, respectively. In the excitation spectrum and the fluorescence emission spectrum, the luminescence intensity of the single strand is the weakest, that of the DNA-DNA strand is slightly stronger, and that of the DNA-RNA strand is the strongest. It can be seen from the figure that compound 114 has only one dye structure in one molecule, and does not form H aggregates, so there is no excitonic effect (in the absorption spectrum, no shift to the short wavelength side is observed). Therefore, compared with compounds with two dye structures, the fluorescence quenching in the single-chain state is weaker, and the fluorescence intensity ratio I _ds /I _ss of the double-chain and single-chain is relatively small. However, due to the flattening of the dye structure by dye intercalation due to duplex formation, as shown in Table 2 below, a greater fluorescence intensity was obtained for the double-stranded state compared to the single-stranded state. In addition, in the single chain, the excitation wavelength in the UV absorption spectrum was changed from 488nm to λ _max (when λ _max is 2, it refers to the long wavelength side), and as a result, the measurement result of quantum yield Φ _F = 0.120 was obtained. In addition, in the DNA-DNA duplex, the excitation wavelength in the UV absorption spectrum was changed from 488nm to λ _max (when λ _max is 2, it refers to the long wavelength side), and as a result, the quantum yield Φ _F = 0.307, double The measurement result of the fluorescence intensity ratio I _ds /I _ss =3.4 of the chain and the single chain.

Table 2

Determination conditions: probe 2.5μM, phosphate buffer 50mM (pH 7.0), NaCl 100mM, complementary chain 2.5μM

The maximum wavelength of fluorescence is a value when excited with light of 488 nm (bandwidth: 1.5 nm).

Quantum yields were calculated using 9,10-diphenylanthracene as a reference.

Example 10

Using the same method as in Example 9, a compound (DNA oligomer) containing only one dye structure in one molecule was synthesized, except that the aforementioned compound 107 used as a dye was replaced by the compound represented by the following chemical formula 115 compound. Synthesis was performed by varying the linker length n from 1 to 4. The sequence is 5'-d(CGCAATXTAACGC)-3' (X is the dye-introducing moiety) identical to the aforementioned compound 105.

Chemical 62

Compound 116 below is the case where n=2. For compound 116, the fluorescence intensity was evaluated in the same manner as in Examples 7-9, and it was found that the fluorescence intensity of the DNA-RNA double strand was increased compared with that of the single strand.

Chemical 63

Fluorescence lifetime measurement

For the DNA oligomer (oligonucleotide) of embodiment 8 (2 dyestuffs) and embodiment 9 (1 dyestuff), under the situation of single strand and under the situation of double-stranded DNA, its fluorescence lifetime is carried out respectively Determination. The assay control DNA oligo contained a dye-introduced nucleotide in position X of the sequence described below.

5'-d(CGCAATXTAACGC)-3' (SEQ ID NO.1)

5′-d(GCGTTAAATTGCG)-3′ (SEQ ID NO.2)

Table 3 below shows the results of the aforementioned fluorescence lifetime measurements. In the table, T is the fluorescence lifetime (ns). CHISQ is the measurement error. T1 represents the time elapsed immediately from the end of the excitation. For the probe containing 2 dyes in Example 8, T2 represents the time elapsed after the elapse of time T1, and for the probe containing 1 dye in Example 9, T2 represents the time elapsed immediately after the end of excitation. T3 represents the time elapsed after the elapse of time T2. In the table, the numerical value represented by "%" is the fluorescence decay rate during the elapsed time T1, T2 or T3 respectively (the fluorescence intensity immediately after the excitation is regarded as 100%), for each probe (DNA oligomer) For , the total amount is 100%. As shown in Table 3, the probe containing 2 dyes (Example 8) has an extremely short quenching process in the single-chain state (0.0210 ns after excitation, the fluorescence decay rate is 81.54%), indicating that there is an excitonic effect. This phenomenon was not observed in other cases. In the single-stranded state of this two-dye-labeled ODN, fluorescence quenching plays an important role in the hybridization specificity and rapid change of fluorescence intensity. In addition, it can be seen from Table 3 that the fluorescence quenching characteristics are consistent with the quadratic or cubic function characteristics. In addition, for the double strands containing 2 dyes in the following table 3, the measurement was carried out again under the same conditions (however, the T1 measurement was omitted), and it was found that the fluorescence decay rate was 44% at T2=2.05, and T3= At 4.38, the fluorescence decay rate was 56%, T=3.33 (ns), CHISQ=1.09, and the values very close to those in Table 3 below were obtained. That is, the probe of this example has good reproducibility in this fluorescence lifetime measurement.

table 3

A dye single strand A dye duplex Two dye single strands Two dye double strands T1 - - 0.0210ns (81.54%) 0.551ns (2.73%) T2 0.934ns (39.19%) 1.58ns (24.63%) 1.28ns (8.99%) 2.33ns (50.30%) T3 3.12ns (60.81%) 3.60ns (75.37%) 3.76ns (9.48%) 4.57ns (46.97%) T 2.26 3.10 0.489 3.33 CHISQ 1.32 0.96 1.11 1.04

chain 2.5 μM

Phosphate buffer 50mM (pH7.0)

NaCl 100mM

Measurements were performed at 455nm (intensification) and 600nm (attenuation).

Example 11

Using the same method as in Example 8, a DNA oligomer represented by the following chemical formula 117 was synthesized, except that the aforementioned compound 107 used as a dye was replaced by a compound represented by the following chemical formula 115'. Compounds with n=3, 4, 5, and 6 can be synthesized in the same manner, respectively. In addition, as in Example 8, this was used as a fluorescent probe, and the performance was evaluated by fluorescence measurement. Table 4 below shows the results thereof. It can be seen from Table 4 that compound 117 has different absorption bands compared with the DNA oligomer (compound 113) of Example 8, but exhibits the same good excitonic effect. This shows that in the present invention, multicolor detection can be performed using fluorescent probes with different absorption bands.

Chemical 64

Chemical 65

Table 4

Example 12

A DNA oligomer (compound 118) represented by the following sequence was synthesized. X is a nucleotide having the same dye structure as in Example 9 (the following formula: chemical formula 118). As shown in the following sequence, this DNA oligomer has two dye-introduced nucleotides arranged consecutively. The introduction of the dye and the synthesis of the DNA oligomer were carried out in the same manner as in the previous examples.

5′-d(TTTTTTXXTTTTTT)-3′ (SEQ ID NO.3)

Chemical 66

In addition, as in the previous examples, this DNA oligomer was used as a fluorescent probe, and the performance was evaluated by fluorescence measurement.

Probe 2.5 μM (chain concentration)

Phosphate buffer 50mM (pH7.0)

NaCl 100mM

Complementary chain 2.5μM (chain concentration)

16 and 17 show the results thereof. Figure 16 is a graph showing the absorption spectrum (the dotted line is the spectrum of the single strand, the solid line is the spectrum of the DNA-DNA chain, and the dotted line is the spectrum of the DNA-RNA chain), and Figure 17 shows the excitation spectrum and fluorescence together A graph of the emission spectrum. In FIG. 17 , the left (short wavelength side) curve represents the excitation spectrum, and the right (long wavelength side) curve represents the fluorescence emission spectrum. In the excitation spectrum and the fluorescence emission spectrum, the luminescence intensity of the single strand is the weakest, that of the DNA-RNA strand is slightly stronger, and that of the DNA-DNA strand is the strongest. As shown in the figure, even when two dye-introduced nucleotides are arranged consecutively in this way, since the distance between the dyes is shortened, an excitonic effect can be exhibited, whereby clear distinction can be made by fluorescence intensity The state of the target nucleic acid before and after hybridization.

Example 13

Compounds (DNA oligomers) represented by the aforementioned chemical formula 113 or 114 with various changes in the linker length n and nucleic acid sequence were synthesized, that is, each ODN shown in Table 5 below. In addition, "ODN" means oligomeric DNA (DNA oligomer) as described above. Carry out synthesis like aforementioned embodiment 1-4, 6, 8, 9 or 12, difference is in order to satisfy the length of linker, change raw material 5-bromopentanoic acid into the compound that carbon atom number (chain length) changes , and appropriately alter the sequence during oligo DNA synthesis. In addition, ODN1 is the same as the oligo DNA (DNA oligomer) synthesized in Example 8, and ODN4 and ODN5 are the same as the oligo DNA (DNA oligomer) synthesized in Example 9. In the synthesis, N-hydroxysuccinimide ester of thiazole orange (compound 109) having 50 equivalents or more of active amino groups was used. After synthesis, development time in reverse HPLC is 20-30 minutes or more as needed. In addition, in the following Table 5, for example, [113] _(n) or [114] _(n) means that the nucleotide represented by the chemical formula 113 or 114 is inserted at the position, and n is the length of the linker. In addition, in Table 5 below, ODN1' indicates a DNA strand complementary to ODN1. Likewise, ODN2' denotes a DNA strand complementary to ODN2, and ODN3' denotes a DNA strand complementary to ODN3.

table 5

Sequence (5'→3')

ODN1 CGCAAT[113] _(n) TAACGC SEQ ID NO.1

ODN1' GCGTTAAATTGCG SEQ ID NO.2

ODN2 TTTTTT[113] ₍₄₎ TTTTTT SEQ ID NO.4

ODN2' AAAAAAAAAAAAA SEQ ID NO.5

ODN3 TGAAGGGCTT[113] ₍₄₎ TGAACTCTG SEQ ID NO.6

ODN3' CAGAGTTCAAAAGCCTTCA SEQ ID NO.7

ODN4 CGCAAT[114] ₍₄₎ TAACGC SEQ ID NO.1

ODN5 CGCAAT[114] ₍₄₎ [114] ₍₄₎ AACGC SEQ ID NO.8

ODN(anti4.5S) GCCTCCT[113] ₍₄₎ CAGCAAATCC[113] ₍₄₎ ACCGGCGTG SEQ ID NO.9

ODN(antiB1) CCTCCCAAG[113] ₍₄₎ GCTGGGAT[113] ₍₄₎ AAAGGCGTG SEQ ID NO.10

As in Example 4 above, the concentration of each ODN synthesized was determined by enzymatic digestion. In addition, each ODN synthesized was identified by MALDI TOF mass spectrometry. The mass analysis values thereof are shown below.

ODN1 (n=3), CGCAAT[113] ₍₃₎ TAACGC, calculated for C ₁₇₈ H ₂₁₃ N ₅₆ O ₇₈ P ₁₂ S ₂ ([MH] ⁺ ) was 4820.7, found 4818.9; ODN1 (n=4 ), CGCAAT[113] ₍₄₎ TAACGC, the calculated value of C ₁₈₀ H ₂₁₇ N ₅₆ O ₇₈ P ₁₂ S ₂ ([MH] ⁺ ) was 4848.8, and the measured value was 4751.4;

ODN1 (n=5), CGCAAT[113] ₍₅₎ TAACGC, calculated for C ₁₈₂ H ₂₂₁ N ₅₆ O ₇₈ P ₁₂ S ₂ ([MH] ⁺ ) was 4876.8, found 4875.6;

ODN1 (n=6), CGCAAT[113] ₍₆₎ TAACGC, calculated for C ₁₈₄ H ₂₂₅ N ₅₆ O ₇₈ P ₁₂ S ₂ (([MH] ⁺ ) 4904.9, found 4903.6;

ODN2, TTTTTT[113] ₍₄₎ TTTTTT, the calculated value of C ₁₈₄ H ₂₂₇ N ₃₄ O ₉₂ P ₁₂ S ₂ ([MH] ⁺ ) is 4822.8, and the measured value is 4821.4;

ODN3, TGAAGGGCTT[113] ₍₄₎ TGAACTCTG, the calculated value of C ₂₅₁ H ₃₀₅ N ₈₁ O ₁₂₄ P ₁₉ S ₂ ([MH] ⁺ ) was 7093.2, and the measured value was 7092.3;

ODN(anti4.5S), GCCTCCT[113] ₍₄₎ CAGCAAATCC[113] ₍₄₎ ACCGGCGTG, the calculated value of C ₃₇₇ H ₄₅₆ N ₁₁₆ O ₁₇₃ P ₂₇ S ₄ ([M.3H] ⁺ ) was 10344.9, measured The value is 10342.7;

ODN(antiB1), CCTCCCAAG[113] ₍₄₎ GCTGGGAT[113] ₍₄₎ AAAGGCGTG, the calculated value of C ₃₈₁ H ₄₅₆ N ₁₂₄ O ₁₇₂ P ₂₇ S ₄ ([M.3H] ⁺ ) was 10489.0, and the found value was 10489.8.

Among the ODNs in Table 5 above, for the ODNs (ODN1, ODN2, ODN3) containing [113](n) with various changes in sequence and linker length, the absorption and excitation spectra were measured before and after hybridization with the complementary strand. and emission spectrum. The results are shown in Table 6 below, Figure 18 and Figure 19 together.

Table 6

Determination conditions: 2.5μM DNA, 50mM sodium phosphate buffer (pH=7.0), 100mM sodium chloride

^b Excited with 488nm

^c is excited by λ _max (when there are two λ _max , use the λ _max on the long wavelength side)

^d Fluorescence intensity ratio of double-stranded state and single-stranded state at λ _em

In addition, in Table 6, ODN1 (n=3-6) has the same structure as the oligomeric DNA (5'-d(CGCAATXTAACGC)-3', X is the dye 113 introduction part) of the aforementioned Example 8. However, in Example 8, the fluorescence quantum yield Φ _F and the fluorescence intensity ratio (I _ds /I _ss ) of the double-strand state and the single-strand state are measured by excitation at a wavelength of 488 nm, while in the present embodiment (Example 13) In, as described above, determined with λ _max excitation in the UV absorption spectrum. Therefore, in the aforementioned Table 1 (Example 8) and the aforementioned Table 6 (Example 13), Φ _F and I _ds /I _ss are different even if the substances are the same.

Fig. 18 is a graph showing the absorption spectrum, excitation spectrum and emission spectrum of ODN containing [113] ₍₄₎ . In each figure (a), (b) and (c), the left figure represents the absorption spectrum, the horizontal axis is the wavelength, and the vertical axis is the absorbance. The graphs on the right represent the excitation spectrum and emission spectrum, the horizontal axis represents the wavelength, and the vertical axis represents the luminous intensity. The ODN containing [113] ₍₄₎ in 50 mM sodium phosphate buffer (pH=7.0) containing 100 mM sodium chloride was used as a sample, and each measurement was performed at 25°C. In each graph of Fig. 18, the black line indicates the measurement result of single-stranded ODN (ss), and the gray line indicates the measurement result of ODN (ds) hybridized with the corresponding complementary strand DNA.

Fig. 18(a) shows the measurement results of ODN1 (n=4) (2.5 µM). The excitation spectrum was measured. For ss, the luminous intensity was measured at a wavelength of 534 nm, and for ds, the luminous intensity was measured at a wavelength of 528 nm. Measure the luminescent spectrum, excite with a wavelength of 519nm for ss, and excite with a wavelength of 514nm for ds.

Fig. 18(b) shows the measurement results of ODN2. The chain concentration is 2.5 μM in the left panel and 1 μM in the right panel. The excitation spectrum was measured. For ss, the luminescence intensity was measured at a wavelength of 534 nm, and for ds, the luminescence intensity was measured at a wavelength of 537 nm. Measure the luminescent spectrum, excite with a wavelength of 517nm for ss, and excite with a wavelength of 519nm for ds.

Fig. 18(c) shows the measurement results of ODN3. The chain concentration was 2.5 μM. Measure the excitation spectrum, for ss, measure the luminous intensity at a wavelength of 535nm, and for ds, measure the luminous intensity at a wavelength of 530nm. Measure the luminescent spectrum, excite with a wavelength of 518nm for ss, and excite with a wavelength of 516nm for ds.

Fig. 19 is a graph showing the absorption spectrum, excitation spectrum and emission spectrum of ODN1 (n=3, 5 and 6). In each of the figures (a), (b) and (c), the left figure shows the absorption spectrum, the horizontal axis is the wavelength, and the vertical axis is the absorbance. Both figures on the right show the excitation spectrum and the emission spectrum, the horizontal axis is the wavelength, and the vertical axis is the luminous intensity. ODN1 (n=3, 5, or 6) in 50 mM sodium phosphate buffer (pH=7.0) containing 100 mM sodium chloride was used as a sample, and each measurement was performed at 25°C. In each graph of FIG. 19 , the black line indicates the measurement result of single-stranded ODN (ss), and the gray line indicates the measurement result of ODN (ds) hybridized with the corresponding complementary strand DNA.

Fig. 19(a) shows the measurement results of ODN1 (n=3) (2.5 µM). The excitation spectrum was measured. For ss, the luminous intensity was measured at a wavelength of 537 nm, and for ds, the luminous intensity was measured at a wavelength of 529 nm. Measure the luminescent spectrum, excite with a wavelength of 521nm for ss, and excite with a wavelength of 511nm for ds.

Fig. 19(b) shows the measurement results of ODN1 (n=5) (2.5 µM). The excitation spectrum was measured. For ss, the luminescence intensity was measured at a wavelength of 538nm, and for ds, the luminescence intensity was measured at a wavelength of 529nm. Measure the emission spectrum, excite with a wavelength of 520nm for ss, and excite with a wavelength of 512nm for ds.

Fig. 19(c) shows the measurement results of ODN1 (n=6). The chain concentration was 2.5 μM. Measure the excitation spectrum, for ss, measure the luminescence intensity at the wavelength of 536nm, and for ds, measure the luminescence intensity at the wavelength of 528nm. Measure the luminescent spectrum, excite with a wavelength of 523nm for ss, and excite with a wavelength of 514nm for ds.

As shown in Table 6, Figure 18 and Figure 19, two absorption bands were observed in the range of 400-550 nm for each ODN sample containing [113] _(n) . When the ODN sample containing 1(n) is in a single-stranded state, the absorption band on the short wavelength side (~480nm) is slightly stronger, while when the ODN sample containing 1(n) is hybridized with the complementary strand, the absorption band on the long wavelength side (~510nm) is slightly stronger. ) absorption bands are prominently (obviously) appearing. The absorption band on the long wavelength side (˜510 nm) is a typical absorption band of a thiazole orange monomer. In the emission spectrum, a single broad absorption band was observed at ~530 nm. ODN samples containing [113] _(n) were hybridized with complementary strands, and the luminescence intensity changed significantly. That is, the [113] _(n) -containing ODN sample hybridized to the target DNA strand exhibits strong fluorescence, while the [113] _(n) -containing ODN sample before hybridization exhibits extremely weak fluorescence compared with after hybridization. In particular, the fluorescence of ODN2 formed by the polypyrimidine sequence is almost completely quenched in the single-chain state. At the maximum luminescence wavelength, the fluorescence intensity ratio (I _ds /I _ss ) of the double chain (double chain) state and the single chain state of ODN2 reaches 160. When ODN3', which is a 20-mer ODN chain, hybridizes with ODN3 of a general sequence, the luminescence intensity is significantly different before and after hybridization. In addition, it can be seen from Table 6, Figure 18(a) and Figure 19 that in ODN1 of this embodiment, when the length of the connector is changed between 3-6, no matter which connector length can obtain a large I _ds /I _ss value. As described above, although the quenching performance of the ODNs shown in Table 6 differed depending on the probe sequence and the length of the linker, any of them showed good quenching performance.

In addition, as shown in the aforementioned Table 6, the melting point (T _m ) of ODN1 (n=4)/ODN1' was increased by 7-9°C compared with the natural double-stranded 5'-CGCAATTTAACGC-3'/ODN1'. This increase in the T _m value indicates that the two cationic dyes in the probe are effectively combined with the duplex formed with the target sequence. In addition, it can be seen from Figs. 18 and 19 that the excitation spectrum has nothing to do with the structure of the compound, but shows a single broad peak around 510 nm. This wavelength was shown to coincide well with one of the absorption bands. That is, it is considered that the absorption related to fluorescence emission is only the absorption band around 510 nm, and the absorption band around 480 nm has little influence on the light emission. In addition, since the absorption band shifts from around 510 nm to around 480 nm due to dye aggregation, the exciton coupling energy is estimated to be 1230 cm ⁻¹ . This is equivalent to the coupling energy reported for H-aggregates of cyanine dyes. However, such theoretical knowledge is not intended to limit the invention.

absorption spectrum

The absorption spectrum of the aforementioned ODN1 (n=4) was measured at various temperatures and concentrations to determine the influence of temperature and concentration on the absorption band. The absorption spectrum in Fig. 20 shows the results. In each of the graphs (a) and (b), the horizontal axis represents the wavelength, and the vertical axis represents the absorbance. Each measurement was performed using ODN1 (n=4) in a 50 mM sodium phosphate buffer (pH=7.0) containing 100 mM sodium chloride as a sample.

Fig. 20(a) shows the change of the absorption spectrum when the temperature of the solution is changed. ODN concentration was 2.5 μM. Spectra were measured at 10°C intervals between 10°C and 90°C.

Fig. 20(b) shows the change of the absorption spectrum when the concentration of the solution is changed. The measurement temperature was 25°C. ODN concentrations were 0.5, 0.75, 1.0, 1.2, 1.5, 2.0, 2.5, 3.0, 4.0 and 5.0 μM.

In addition, the insert is a graph showing the relationship between the logarithm of absorbance at a wavelength of 479 nm (vertical axis) and the logarithm of absorbance at a wavelength of 509 nm (horizontal axis).

As shown in Fig. 20(a), when the temperature of the sample was changed and the measurement was performed, it was found that the absorbance ratio of the two absorption bands slightly changed. That is, as the sample temperature rises, the absorption band at 479 nm gradually decreases and the absorption band at 509 nm increases. However, it can be seen from the figure that this change is extremely weak. This indicates that the structure of the probe ODN1 (n=4) of the present invention changes very slightly with temperature, and thus can be used almost independently of temperature. In addition, as shown in the figure, at 487 nm, an isosbestic point indicating the presence of two spectral constituents was observed.

On the other hand, as shown in FIG. 20( b ), when the sample concentration of ODN1 (n=4) was increased, an increase in the absorbance of the absorption bands on both sides was observed. In addition, as shown in the inset, the graph of log(Abs ₄₇₉ ) versus log(Abs ₅₀₉ ), that is, the ratio of the logarithm of the absorbance of each absorption band, represents a straight line. This shows that the ratio of the components representing the two spectra is almost constant regardless of the ODN concentration. That is, even if the concentration in the solution is changed, the structure of the probe ODN1 (n=4) of the present invention hardly changes, and thus can be used without being affected by the concentration.

In addition, the reasons for the spectrum changes in Fig. 20 (a) and (b) can be explained as follows, but these explanations are an example of theoretical understanding and do not limit the present invention. That is, first, ODN1 (n=4) formed intramolecular H aggregates through the dichroic system. It is speculated that the spectral change in Fig. 20(a) is due to the slight loosening of the structure of the H aggregates due to the temperature increase. In addition, it is considered that the formation of the aforementioned intramolecular H aggregates ends in the molecule, so even if the concentration increases, there is almost no structural change caused by intermolecular interactions, so as shown in Figure 20(b) and the illustration, the two spectra The ratio of the constituent elements is almost constant. In addition, it is considered that in the sample solution of ODN1 (n=4), there are two conformational models of intramolecular H aggregates and dye monomers (dye moieties are not aggregated). It is presumed that the absorption band on the short wavelength side (479 nm) comes from intramolecular H aggregates. Since the absorption band (509 nm) on the long wavelength side increases by heating, it is presumed to be derived from the dye monomer.

CD Spectrum

The CD spectrum of ODN1 (n=4)/ODN1' was determined. The chain concentration was 2.5 μM, measured at 25° C. in 50 mM sodium phosphate buffer (pH=7.0) containing 100 mM sodium chloride. The CD spectrum in Fig. 21 shows the measurement results. In this figure, the horizontal axis represents the wavelength (nm), and the vertical axis represents the angle θ. As shown in the figure, the ODN1 (n=4)/ODN1' double strand exhibits a split-Cotton effect at 450-550 nm. That is, the measured CD pair exhibits a typical pattern when the thiazole orange dye is inserted into a DNA double strand. That is, it is considered that the dye portion of ODN1 (n=4) is inserted into the formed double-stranded DNA, thereby greatly hindering the formation of dichroic aggregates (H aggregates). This CD measurement result together with the aforementioned Tm measurement result indicated that when the dye moiety in ODN1 (n=4) binds to double-stranded DNA, both dye moieties are inserted into the major groove, thereby forming a thermostable double-stranded structure. However, this theoretical understanding does not limit the invention. The resulting double strands are thermally stable, which indicates that the probe (nucleic acid) of the present invention can be effectively used in the detection of complementary sequences.

Example 14

For the aforementioned ODN5 (CGCAAT[114] ₍₄₎ [114] ₍₄₎ AACGC), the absorption spectrum, excitation spectrum and emission spectrum of double-chain state and single-chain state were measured. Table 7 below and Figure 22 show the results.

Table 7

Determination conditions: 2.5μM DNA, 50mM sodium phosphate buffer (pH=7.0), 100mM sodium chloride

^b Excited with 488nm

^c is excited by λ _max (when there are 2 λ _max , use λ _max on the long wavelength side)

^d Fluorescence intensity ratio of double-stranded state and single-stranded state at λ _em

Figure 22 is a graph showing the absorption spectrum, excitation spectrum and emission spectrum of ODN5, an ODN containing [114] ₍₄₎ . The chain concentration of ODN5 was 2.5 μM, and it was measured at 25° C. in 50 mM sodium phosphate buffer (pH=7.0) containing 100 mM sodium chloride. The black line indicates the measurement results of single-stranded ODN5(ss), and the gray line indicates the measurement results of double-stranded ODN5(ds) hybridized with ODN1'. (a) is the absorption spectrum, the horizontal axis is the wavelength, and the vertical axis is the absorbance. (b) represents the excitation spectrum (curve on the short-wavelength side) and emission spectrum (curve on the long-wavelength side), the horizontal axis represents the wavelength, and the vertical axis represents the emission intensity. The excitation spectrum was measured. For ss, the luminous intensity was measured at a wavelength of 534 nm, and for ds, the luminous intensity was measured at a wavelength of 514 nm. Measure the luminescent spectrum, excite with a wavelength of 528nm for ss, and excite with a wavelength of 519nm for ds.

As shown in the aforementioned Table 7 and Figure 22, compared with the luminescence inhibition of single-stranded ODN4 containing only one [114] ₍₄₎ nucleotide (Table 2 of the aforementioned Example 9), two [114] ₍₄₎ The sequence ODN5 in which nucleotides are linked together exhibits more efficient fluorescence quenching. In the absorption spectrum of ODN5 in the single-chain state, the absorption band shifts toward the short wavelength side. This indicated that two [114] ₍₄₎ nucleotides contained in ODN5 formed an intramolecular H aggregate. This aggregation triggered quenching of single-chain ODN5 as observed in [113] _(n) -containing ODNs. In other words, it is considered that due to the H aggregation of the dye moiety of the two [114] ₍₄₎ nucleotides in ODN5, exciton coupling occurs between the aforementioned dyes, which is the cause of fluorescence emission suppression (quenching) where. This indicated that, like the ODN containing [113] _(n) , ODN5 containing 2 [114] ₍₄₎ nucleotides could be used in complementary strand detection.

Example 15

Fluorescence of ODN1 (n=4) hybridized to complementary ODN1' was determined visually. Fig. 23 shows the measurement results thereof. In this figure, the sample tank on the left is the sample tank equipped with ODN1 (n=4) single strand, and in this figure, the sample tank on the right is the sample tank equipped with ODN1 (n=4)/ODN1′ double strand , respectively show the state after irradiation with a 150W halogen lamp. The chain concentration in each sample well was 2.5 µM, and 50 mM phosphate buffer (sodium phosphate buffer) (pH 7.0) and 100 mM NaCl were filled. As shown in the figure, after being irradiated by a 150W halogen lamp, the sample chamber on the left side of the figure containing ODN1(n=4) single strands had almost no fluorescence, while the sample chamber on the right side of the figure containing ODN1(n=4)/ODN1′ double strands The side sample wells show a very pronounced pale green fluorescence. In addition, the same result was obtained by replacing the complementary DNA strand ODN1' with the corresponding complementary RNA strand. In addition, the same results were also obtained in ODN2 and ODN2'. In addition, for ODN2 and ODN2', the same result was obtained by replacing ODN2' with the corresponding complementary RNA (A13mer). Also, in these cases, the chain concentration was 5 μM. In addition, the same results were also obtained for all other ODNs in the aforementioned Table 6. Therefore, with the ODN of this example, the fluorescence intensity changes significantly due to hybridization, and therefore, the target sequence that may hybridize can be easily judged by naked eyes. This suggests that these ODNs can be used for intuitive genetic analysis.

Example 16

As in Example 8, a DNA oligomer represented by the following chemical formula 120 was synthesized, except that the aforementioned compound 107 used as a dye was replaced by a compound represented by the following chemical formula 119.

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Compounds (oligomeric DNA) having n=3, 4, 5 and 6 in the above formula 120 can be synthesized in the same manner, respectively. In addition, using an ODN represented by the sequence 5'-d(CGCAAT[120] ₍₅₎ TAACGC)-3' (which is ODN6 (n=5)) as a fluorescent probe, the absorption spectrum and fluorescence emission spectrum were measured and performance was evaluated. Measuring conditions are the same as in Example 7. Fig. 24 shows the measurement results. Fig. 24(a) is the absorption spectrum, the horizontal axis represents the wavelength (nm), and the vertical axis represents the absorbance. Fig. 24(b) is the fluorescence emission spectrum, the horizontal axis represents the wavelength (nm), and the vertical axis represents the emission intensity. In each panel, the black line represents the spectrum of single-stranded ODN, and the gray line represents the spectrum of double-stranded ODN hybridized with complementary ODN. As shown in FIG. 24( a ), in the double-stranded ODN, due to the formation of a double helix, the maximum wavelength of UV absorption around 600 nm shifts toward the longer wavelength side. In addition, as shown in Fig. 24(b), in the double-stranded ODN, the fluorescence intensity was greatly increased compared with the single-stranded one. Therefore, it is considered that the exciton effect is exhibited in the single-chain state. That is to say, although the ODN (compound 120) of the present embodiment has different absorption bands compared with the ODN (compound 113) of embodiment 8 and the ODN (compound 117) of embodiment 11, it can also show good excitonic effect. This shows that in the present invention, multicolor detection is possible using fluorescent probes with different absorption bands.

Example 17: Forming a double strand with RNA

In a cuvette, make the aforementioned ODN2 (sequence 5′-d(TTTTTT[113] ₍₄₎ TTTTTT)-3′) and its corresponding complementary RNA strand (RNA A13 polymer) form a double-stranded ODN, and determine Fluorescence spectrum. Also, add RNase H to it and watch how the spectrum changes. Fig. 25 shows the results. In this figure, the horizontal axis represents time, and the vertical axis represents fluorescence intensity. In the figure, the black line represents the spectral change of the aforementioned double-stranded ODN with RNase H added midway, and the gray line represents the spectral change of the control, that is, the aforementioned double-stranded ODN without RNase H added. Measurement was performed using the aforementioned spectrofluorophotometer while stirring at 37°C. As shown in the figure, if RNase H is added, the RNA hybridized with the aforementioned ODN2 will be digested, and the aforementioned ODN2 will return to a single strand, so the fluorescence intensity will gradually decrease. This fact also indicates that the probe (nucleic acid) of the present invention can be used to detect complementary RNA by fluorescence.

Example 18

Change the concentration ratio of the complementary DNA strand ODN1' (sequence 5'-d(GCGTTAAATTGCG)-3') of the aforementioned ODN1 (n=4) (sequence 5'-d(CGCAAT[113] ₍₄₎ TAACGC)-3') To observe the change of fluorescence intensity. The measurement conditions were that the chain concentration of ODN1 (n=4) was fixed at 1.0 μM, the phosphate buffer was 50 mM (pH 7.0), NaCl was 100 mM, and the excitation wavelength was 488 nm (bandwidth: 1.5 nm). The assay was performed at each concentration of the complementary chain ODN1' at 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 or 3.0 µM. Fig. 26 shows the measurement results. In this figure, the horizontal axis represents the equivalent number of ODN1' to ODN1 (n=4). The vertical axis represents the fluorescence emission intensity (relative value) at the fluorescence λ _max (529 nm). As shown in the figure, when the equivalence number of ODN1' is less than 1, the fluorescence emission intensity exhibits a direct proportional relationship with respect to the above-mentioned equivalence number with extremely high accuracy, but when the above-mentioned equivalence number exceeds 1, there is no change. This indicated that ODN1 (n=4) hybridized correctly with ODN1' at a 1:1 mass ratio (molecule number ratio).

As described above, when the amount of ODN1' (target DNA) is the same as or less than the amount of ODN1 (n=4) (probe), the fluorescence intensity increases in proportion to the concentration of the target DNA. That is, in a system where ODN1' (target DNA) exists, if an excess amount of ODN1 (n=4) (probe) is added, the aforementioned target DNA can be quantified by fluorescence intensity measurement. In addition, by tracking the increase and decrease of the aforementioned fluorescence intensity, the aforementioned increase and decrease of the target DNA can also be measured.

In order to quantify the aforementioned target DNA in the system, for example, a standard curve may be prepared in advance as shown in FIG. 26 . For example, if the fluorescence intensity of a sample with an unknown concentration of ODN1' (target DNA) is 80 when measured under the same conditions as in this example, then it can be seen from Figure 26 that the concentration of the aforementioned ODN1' (target DNA) is about 0.55 μM .

In fact, by quantifying the ODN1' (target DNA) sequence in the nucleic acid by the above-mentioned method, while immediately detecting the occurrence of phenomena such as amplification, decomposition, and protein binding of the target sequence, it is also possible to quantify the amount of these phenomena. Quantitative.

Example 19: Dot blot analysis

In order to observe the changes in fluorescence properties of the new probe (nucleic acid) synthesized at this time due to hybridization, DNA was analyzed by dot blotting using the aforementioned ODN (antiB1) and ODN (anti4.5S). The target DNA sequence used is a short-chain DNA fragment containing the B1 RNA sequence. This sequence is one of the short scattered intranuclear repeats in the rodent genome. In addition, the aforementioned short-chain DNA fragments contain 4.5S RNA sequences. This sequence is one of the low-molecular-weight nuclear RNAs isolated from rodent cells, and has extensive homology with the B1 family. In this example, by preparing imprinted probes ODN (antiB1) and ODN (anti4.5S), and introducing 2 [113] ₍₄₎ nucleotides into them, high sensitivity and high fluorescence intensity can be imparted . In addition, the structures of ODN (antiB1) and ODN (anti4.5S) are as described in Table 5 in Example 13 above.

More specifically, the dot blot analysis of this example was performed as follows. That is, first, the following two DNA fragments (1) and (2) were prepared by the aforementioned automatic DNA synthesizer.

(1) The following DNA double strands containing 4.5S RNA sequence and its complementary DNA.

5′-d(GCCGGTAGTGGTGGCGCACGCCGGTAGGATTTGCTGAAGGAGGCAGAGGCAGGAGGATCACGAGTTCGAGGCCAGCCTGGGCTACACATTTTTTT)-3′ (SEQ ID NO. 11)

(2) The following DNA duplexes containing the B1 RNA sequence and its complementary DNA.

5′-d(GCCGGGCATGGTGGCGCACGCCTTTAATCCCAGCACTTGGGAGGCAGAGGCAGGCGGATTTCTGAGTTCGAGGCCAGCCTGGTCTACAGAGTGAG)-3′ (SEQ ID NO. 12)

The aforementioned DNA double strands were denatured in an aqueous solution containing 0.5M sodium hydroxide and 1M sodium chloride. An aliquot of this denatured DNA was subjected to dot (dot) blotting on a positively charged nylon membrane (Roche). After this positively charged nylon membrane was wetted with an aqueous solution containing 0.5M sodium phosphate and 1M sodium chloride, it was incubated at 50°C for 30 minute. Subsequently, the aforementioned positively charged nylon membrane was incubated at 50° C. for 1 hour in an aqueous probe solution containing 0.5 M sodium phosphate and 1 M sodium chloride (the probe was 150 pmol of ODN (anti4.5S) or ODN (antiB1)). After it was cooled at room temperature, the hybridization buffer was removed, new phosphate buffer was added, and the fluorescence emitted by the aforementioned positively charged nylon membrane was observed by BioRad's VersaDoc imaging system (trade name). The excitation light used was light obtained by passing light emitted from Wakenyaku Co., Ltd. UV transilluminator Model-2270 (trade name) through a UV/cyan light conversion plate (UVP).

Fig. 27 shows the measurement results.

Fig. 27(a) is a schematic view showing the state of Southern blotting of different sequences on a nylon membrane. The 4 dots in the upper row represent DNA containing 4.5S RNA sequence, and the 4 dots in the lower row represent DNA containing B1 RNA.

Fig. 27(b) is a graph showing fluorescence emission after incubation in a solution containing ODN (anti4.5S).

Fig. 27(c) is a graph showing fluorescence emission after incubation in a solution containing ODN (antiB1).

As shown in FIG. 27 , the fluorescence of blotted spots can be read at room temperature with a fluorescence imaging device without repeated washing after blot analysis. When ODN (anti4.5S) was added, as a result of incubation with the aforementioned probes, strong fluorescence was obtained from the spots of the 4.5S sequence, but the fluorescence from the spots of the B1 sequence was negligible. In contrast, when ODN (antiB1) was added, the B1 spot showed strong fluorescence, but only extremely weak fluorescence was observed at the 4.5S spot. Thus, the probes of the present invention enable analyzes significantly different from previous blot assays without the need for tedious multi-step washes after blot and without the need for antibody or enzyme treatment. In addition, unlike on-off probes such as molecular beacons, the probe of the present invention is easy to introduce multiple fluorescent dye-labeled parts, so that the fluorescence intensity can be further enhanced. This is a very advantageous point of the present invention. The aforementioned fluorescent dye labeling moiety may also be included in the [113] ₍₄₎ nucleotide as described in this example, for example.

Example 20

The poly-T probe containing the dye of linker length n=4 in Example 8 (the aforementioned ODN2) was introduced into the cells by microinjection using a glass microtube, and passed through a cell equipped with a mercury lamp, a cooled CCD camera, and a fluorescence filter An inverted microscope of the device (for YFP) was used to measure fluorescence luminescence. 28 to 30 show the results. Fig. 28 is a photo of differential interference, Fig. 29 is a photo of fluorescence observation, and Fig. 30 is a superimposed view of Fig. 28 and Fig. 29 . As shown in the figure, the fluorescent probe (label) of the present invention binds to the poly A-terminal sequence of the mRNA expressed in the cell to emit light. In other words, the fluorescent probe (label) of the present invention can effectively detect genes not only in vitro, but also in vivo.

Example 21

In addition, the common fluorescent dye Cy5 is combined with the aforementioned ODN2 (sequence 5'-d(TTTTTT[113] ₍₄₎ )TTTTTT)-3') by conventional methods, and then introduced into cells by the aforementioned methods. Here, Cy5 is combined with it by being added to the 5' end of the aforementioned ODN2 by an automatic DNA synthesizer during the synthesis of the aforementioned ODN2 (sequence 5'-Cy5-d(TTTTTT[113] ₍₄₎ TTTTTT)-3 '). Fluorescence luminescence was measured by laser scanning confocal microscopy. Fig. 31 shows the results. Fig. 31A shows the fluorescence above 650 nm obtained by excitation at 633 nm, which is the fluorescence emitted by Cy5. Figure 3 IB shows the fluorescence at 505-550 nm obtained with excitation at 488 nm, which is the fluorescence emitted by the 2 thiazole orange moieties. As shown, ODN2 binds to the poly-A-terminal sequence of mRNA expressed in cells to emit light. Thus, the distribution of mRNA within cells can be traced. Various kinds of dyes (atomic groups exhibiting fluorescence) can also be introduced into the compound or nucleic acid of the present invention in this manner. In this way, for example, multicolor detection can be performed due to the difference in the fluorescence λ _max of each dye.

Example 22

The aforementioned ODN2 (sequence 5′-d(TTTTTT[113] ₍₄₎ TTTTTT)-3′) was introduced into the nucleus by the aforementioned method, and after the injection was completed (after 0 seconds) to about 4 and a half minutes later, a total of Fluorescence emission was tracked by focusing microscope (excitation at 488nm, fluorescence at 505-550nm). Fig. 32 shows the results. In this figure, it is divided into 11 panels, from left to right, and from top row to bottom row, showing the passage after ODN2 injection. In each figure, the elapsed time (after ODN2 injection) is shown in Table 8 below. As shown in the figure, it was indicated that the probe ODN2 was concentrated in the nucleus immediately after injection, but was gradually dispersed throughout the cell while hybridizing with mRNA (poly A). mRNA can be tracked in this way by the present invention.

【Table 8】

0 seconds 8 seconds 38 seconds 68 seconds 98 seconds 128 seconds 158 seconds 188 seconds 218 seconds 248 seconds 278 seconds -

Example 23

ODNs were synthesized in which the Ts on both sides of [113] ₍₄₎ in the aforementioned ODN2 were increased to 24, respectively. Call it ODN7. The synthesis was carried out in the same way as the synthesis of ODN2. In addition, the sequence of ODN7 is

5'-d(TTTTTTTTTTTTTTTTTTTTTTTT[113] ₍₄₎ TTTTTTTTTTTTTTTTTTTTTTTT)-3') (SEQ ID NO. 13). It was injected into the nucleus in the same manner as in Example 22, and the fluorescence intensity was measured. Fig. 33 shows fluorescent photographs after a certain period of time. ODN7 was concentrated in the nucleus immediately after injection, but as in Example 22, it was gradually dispersed throughout the cell while hybridizing with mRNA (poly A), and finally, as shown in Figure 33, it showed a pattern of cells scattered around the nucleus. state.

Example 24: Multicolor detection

As described in Examples 11, 16, etc., by changing the absorption wavelength, emission wavelength, etc., the fluorescent probe of the present invention can perform multicolor complementary chain detection. This multicolor detection can be achieved, for example, by changing the structure of the dye (atomic group exhibiting fluorescence) as in the aforementioned compounds 113, 117 and 120. In this example, multiple types of fluorescent probes were synthesized (manufactured), and multicolor complementary strand detection was carried out

First, a DNA chain (probe) having a nucleotide structure represented by the following formula (121) is synthesized by variously changing the structure of the dye (atomic group exhibiting fluorescence). In the following formula (121), "Dye" represents a dye moiety.

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Specifically, compounds (DNA strands) 113, 120, 122, 123 and 124 in which "Dye" moieties in the aforementioned formula (121) are represented by the following formulas, respectively, were synthesized. n is the linker length (number of carbon atoms) respectively. For compounds 113, 120, 122, 123 and 124, compounds with n=3, 4, 5 and 6 were synthesized, respectively. The synthesis method is the same as that of the aforementioned Examples 1-4, 6, 8, 9, 12, 13 or 16, except that the aforementioned dye 107 is replaced by a dye with a corresponding structure. Synthesis of a dye that replaces the aforementioned dye 107 can also be performed like the synthesis of the aforementioned dye 107 (Scheme 5 of the previous embodiment 6), except that the structure of the raw material is appropriately changed. In addition, compounds 113 and 120 have the same structures as compounds 113 and 120 in the foregoing examples, respectively.

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For the above compounds 113, 120, 122, 123 and 124, ODNs represented by the sequence 5'-d(CGCAATX _(n) TAACGC)-3' were synthesized, respectively. X is 113, 120, 122, 123 or 124. n is the linker length. The ODN represented by the sequence 5'-d(CGCAAT[113] _(n) TAACGC)-3' is the same as the aforementioned ODN1. The ODN represented by the sequence 5'-d(CGCAAT[120] _(n) TAACGC)-3' is the same as the aforementioned ODN6. The ODN represented by the sequence 5'-d(CGCAAT[122] _(n) TAACGC)-3' is ODN8. The ODN represented by the sequence 5'-d(CGCAAT[123] _(n) TAACGC)-3' is ODN9. The ODN represented by the sequence 5'-d(CGCAAT[124] _(n) TAACGC)-3' is ODN10. For ODN1, ODN6, ODN8, ODN9 and ODN10, ODNs with n=3, 4, 5 and 6 were synthesized, respectively.

For ODN1 (n = 4), ODN6 (n = 4), ODN8 (n = 4), ODN9 (n = 4) and ODN10 (n = 4), the fluorescence was measured after forming a double strand with the complementary strand ODN1', respectively emission spectrum. The measurement conditions other than the excitation wavelength are the same as those in the previous examples. The results are shown in Table 9 below. In Table 9 below, _Ex represents the excitation wavelength, and E _m represents the maximum wavelength of fluorescence emission. In addition, the excitation wavelength _Ex is almost equal to the absorption maximum wavelength λ _max .

Table 9

Double-stranded structure E _x E _m

5'-d(CGCAAT[113] ₍₄₎ TAACGC-3'(ODN1(n=4)/ODN1' 514nm 528nm

5'-d(CGCAAT[120] ₍₄₎ TAACGC-3'(ODN6(n=4)/ODN1' 650nm 654nm

5'-d(CGCAAT[122] ₍₄₎ TAACGC-3'(ODN8(n=4)/ODN1' 436nm 456nm

5'-d(CGCAAT[123] ₍₄₎ TAACGC-3'(ODN9(n=4)/ODN1' 534nm 550nm

5'-d(CGCAAT[124] ₍₄₎ TAACGC-3'(ODN10(n=4)/ODN1' 541nm 563nm

It can be seen from Table 9 that in the wide wavelength range of 456nm-654nm, each ODN exhibits different maximum wavelengths E _m of fluorescence emission when forming double strands. That is, using the ODN synthesized in this example (Example 24), it is possible to perform multicolor complementary strand DNA detection. In addition, for the compounds (DNA strands) 113, 120, 122, 123 and 124, ODN1, ODN6, ODN8, ODN9 and ODN10 of this example, it was determined that they can be used in the same way as the previous examples, and all of them can be complemented in a multicolor manner. Strand RNA detection, dot blot analysis, intracellular mRNA detection, etc.

Possibility of industrial use

As can be seen from the above, the present invention provides a label capable of efficiently detecting, for example, a nucleic acid double helix structure. In addition, the present invention provides nucleic acid detection methods and kits using the aforementioned markers. The compounds or nucleic acids of the present invention, the aforementioned compounds of the present invention and nucleic acids have the aforementioned formulas (1), (1b), (1c), (16), (16b), (17), (17b), (18) Or the characteristic structure shown in (18b), thus can be used, for example, as a marker for effectively detecting the nucleic acid double helix structure. The marker of the present invention is excellent in nucleic acid detection sensitivity, and thus can be used in a wide range of applications such as research, clinical use, diagnosis, in vitro gene detection, and in vivo gene detection. In addition, there is no limitation on the use of the compound and nucleic acid of the present invention, and any use is acceptable.

Claims (11)

1. A nucleic acid or a salt thereof comprising at least one structure represented by the following formula (16), (16b), (17) or (17b),

In equations (16), (16b), (17) and (17b), B is an atomic group having the following structure: a natural nucleic acid base skeleton selected from the following: adenine skeleton, guanine skeleton, cytosine skeleton, thymine skeleton and uracil skeleton, or an artificial nucleic acid base skeleton selected from the following: Py , Py der., Pu and Pu der., in, The Py is an atomic group having a covalent bond with E at the 1-position and a covalent bond with the linker part at the 5-position in the six-membered ring represented by the following formula (11), The Py der. is an atomic group in which at least one of all the atoms of the six-membered ring of Py is replaced by an N, C, S or O atom, and the N, C, S or O atom may optionally have a charge, hydrogen atoms or substituents, The Pu is an atomic group having a covalent bond with E at the 9-position and a covalent bond with the linker part at the 8-position in the condensed ring represented by the following formula (12), The Pu der. has at least one atomic group replaced by an N, C, S or O atom in all the atoms of the five-membered ring of the Pu, and the N, C, S or O atom can optionally have a charge, hydrogen atom or substituent,

Z ¹¹ and Z ¹² respectively represent fluorescent atomic groups exhibiting excitonic effects, Each of L ¹ , L ² and L ³ is a linker, and the length of the main chain is arbitrary. In the main chain, each can contain or not contain C, N, O, S, P and Si. In the main chain, each can be Containing or not containing single bond, double bond, triple bond, amide bond, ester bond, disulfide bond, imine group, ether bond, thioether bond and thioester bond, L ¹ , L ² and L ³ can be mutually same or different, D is CR, N, P, P=O, B or SiR, R is a hydrogen atom or an alkyl group, b is a single bond, double bond or triple bond, Alternatively, in the formulas (16) and (16b), L ¹ and L ² are the linkers, L ³ , D and b do not exist, and L ¹ and L ² are directly connected to B, where, in the formulas (16) and (17), E is an atomic group having a deoxyribose skeleton, a ribose skeleton or a structure derived from any of these skeletons, at least one O atom in the phosphate bridge can be replaced by an S atom, In equations (16b) and (17b), E is an atomic group having a peptide structure or a peptidomimetic structure, and In formulas (17) and (17b), each B may be the same or different, and each E may be the same or different. 2. The nucleic acid or salt thereof according to claim 1, wherein in said formulas (16) and (17), E is an atomic group having a backbone structure of DNA, modified DNA, RNA, modified RNA or LNA, where In the above formulas (16b) and (17b), E is an atomic group having a main chain structure of PNA. 3. The nucleic acid or salt thereof according to claim 1, wherein in said formulas (16), (16b), (17) and (17b), the main chain lengths of L ¹ , L ² and L ³ are each 2 above integer. 4. The nucleic acid or salt thereof according to claim 1, wherein Z ¹¹ and Z ¹² are each independently derived from thiazole orange, oxazole yellow, cyanine, hemicyanine, other cyanine dyes, methyl red, azo A monovalent group of a dye or its derivative. 5. The nucleic acid or salt thereof according to claim 1, wherein Z ¹¹ and Z ¹² are each independently an atomic group represented by any one of the following formulas (7)-(9):

Among them, in formulas (7)-(9), X ¹ and X ² are each S or O, which can be the same or different, n is 0 or a positive integer, R ¹ -R ¹⁰ , R ¹³ -R ²¹ are each independently a hydrogen atom, a halogen atom, a straight chain or branched alkyl group with 1-6 carbons, a straight chained or branched chain alkoxy group with 1-6 carbons base, nitro or amino, Among R ¹¹ and R ¹² , one is the same as L ¹ or L ² in the formula (16), (16b), (17) or (17b), or the formula (16-1), (16-2 ), (16b-1), (16b-2), (17-1) or (17b-1) in the NH-bonded linking group, the other is a hydrogen atom or a direct carbon number of 1-6 chain or branched chain alkyl, Where multiple R ¹⁵ exist in formula (7), (8) or (9), they may be the same or different, Where multiple R ¹⁶ exist in formula (7), (8) or (9), they may be the same or different, X ¹ , X ² and R ¹ to R ²¹ in Z ¹¹ and X ¹ , X ² and R ¹ to R ²¹ in Z ¹² may be the same or different from each other. 6. The nucleic acid or salt thereof according to claim 5, wherein in formulas (7)-(9), among R ¹¹ and R ¹² , the linking group is polymethylenecarbonyl having 2 or more carbon atoms , through the carbonyl moiety and L ¹ or L ² in the formula (16), (16b), (17) or (17b), the formula (16-1), (16-2), (16b-1 ), (16b-2), (17-1) or NH bonding in (17b-1). 7. The nucleic acid or salt thereof according to claim 1, wherein in said formulas (16), (16b), (17) and (17b), B is said Py, said Py der., said Pu or the Pu der. 8. A label, wherein two planar chemical structures in a molecule are not in the same plane, but exist at an angle, but when the molecule is inserted or grooved into a nucleic acid, the two planar chemical structures Arranged in the same plane to produce fluorescent light, Wherein the label is formed by two or more groups of dye molecules, wherein although the excitonic effect due to the parallel aggregation of two or more dye molecules does not exhibit fluorescence emission, these molecules bind to the nucleic acid when intercalated or grooved. In the middle, the release of the aggregation state will produce fluorescence emission, Wherein said marker is: The nucleic acid or salt thereof according to claim 1, wherein each of Z ¹¹ and Z ¹² is a fluorescent atomic group exhibiting an excitonic effect. 9. A complex marker having a chemical structure having two or more dye molecules in the same molecule as a characteristic chemical structure, wherein although the excitonic effect due to the parallel aggregation of two or more dye molecules does not Exhibits fluorescence, but these molecules fluoresce upon intercalation or groove binding into a nucleic acid by release of said assembled state, The complex marker optionally has a structure in which two or more dye molecules are bound to linker molecules bound to the nucleic acid to be labeled, the binding of the dye molecules to the linker molecules being via additional links Linker molecules to form branched structures, or direct bonding without the aid of additional linker molecules, Wherein optionally, each of the dye molecules is the molecule described in claim 8. 10. A marker, which is a marker mononucleotide, a marker oligonucleotide, a marker nucleic acid or a marker nucleic acid analogue, wherein the marker is composed of the marker according to claim 8, or has an exciton effect The nucleic acid or salt labeling of claim 1 of the fluorescent atomic groups Z ¹¹ and Z ¹² , the nucleic acid optionally has at least one base contained in a mononucleotide, an oligonucleotide, a nucleic acid or a nucleic acid analog A linker molecule bonded to the 5-carbon atom of the pyrimidine nucleus or the 8-position carbon atom of the purine nucleus. 11. A kit comprising a nucleic acid synthesis device, a label and a fluorescence intensity measurement device, wherein the label is the label according to claim 8.

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